Novel Anti-Human Transferrin Receptor Antibody Capable of Penetrating Blood-Brain Barrier

ABSTRACT

Provided is an anti-human transferrin receptor antibody or an analog thereof, wherein in the heavy chain variable region of the antibody, (a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 or SEQ ID NO: 14, and (c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 or SEQ ID NO: 16, and an analogue thereof.

TECHNICAL FIELD

The present invention relates to an anti-human transferrin receptorantibody to be utilized for conjugation with a compound that needs toexhibit its function in the central nervous system when administeredparenterally (a protein or a low-molecular-weight compound and the like)in order to make that compound able to pass through the blood-brainbarrier after parenterally administered, and also a method forproduction thereof, as well as a method of use thereof.

BACKGROUND ART

Unlike the capillaries in other tissues such as muscles, the capillariesthat supply the blood to most of the brain tissues except some areasincluding the circumventricular organs (pineal gland, pituitary body,area postrema, etc.) differ in that the endothelial cells forming theirendothelium are mutually connected by tight intercellular junctions.Passive transfer of substances from the capillaries to the brain isthereby restricted, and although there are some exceptions, substancesare unlikely to move into the brain from the blood except such compoundsas are lipid-soluble or of low-molecular-weight (less than 200-500Dalton) and electrically neutral around the physiological pH. Thissystem, which restricts exchange of substances between the blood and thetissue fluid of the brain through the endothelium of capillaries in thebrain, is called the blood-brain barrier or BBB. The blood-brain barriernot only restricts exchange of substances between the blood and thebrain but also between the tissue fluid of the central nervous system,including the brain and the spine, and the blood.

Owing to the blood-brain barrier, most of the cells of the centralnervous system escape the effects of fluctuating concentrations ofsubstances like hormones and lymphokines in the blood, and theirbiochemical homeostasis is thus maintained.

The blood-brain barrier, however, imposes a problem when it comes todevelop pharmaceutical agents. For mucopolysaccharidosis type I (Hurlersyndrome), an inherited metabolic disease caused by α-L-iduronidasedeficiency, for example, although an enzyme replacement therapy iscarried out by intravenous supplementation with a recombinantα-L-iduronidase as a therapy, the therapy is not effective for thenotable abnormality observed in the central nervous system (CNS) inHurler syndrome because the enzyme cannot pass through the blood-brainbarrier.

Development of various methods has been attempted to make thosemacromolecular substances as proteins or the like, which need to bebrought into function in the central nervous system, pass through theblood-brain barrier. In the case of nerve growth factor, for example,while attempts have been made for a method to cause the factor to passthrough the blood-brain barrier by allowing liposomes encapsulating thefactor to fuse with the cell membrane of endothelial cells in braincapillaries, they have not been materialized (Non Patent Literature 1).In the case of α-L-iduronidase, an attempt has been made to enhance thepassive transfer of the enzyme through the blood-brain barrier byraising its blood concentration through an increased single dose of theenzyme, and it thus has been demonstrated, using a Hurler syndromeanimal model, that the abnormality in the central nervous system (CNS)is ameliorated by that method (Non Patent Literature 2).

Furthermore, circumventing the blood-brain barrier, an attempt has alsobeen made to administer a macromolecular substance directly into themedullary cavity or into the brain. For example, reports have been madeabout a method in which human α-L-iduronidase was administered into themedullary cavity of a patient with a Hurler syndrome(mucopolysaccharidosis type I) (Patent Literature 1), a method in whichhuman acid sphingomyelinase was administered into the brain ventriclesof a patient with Niemann-Pick disease (Patent Literature 2), and amethod in which iduronate 2-sulfatase (I2S) was administered into thebrain ventricles of Hunter syndrome model animals (Patent Literature 3).While it seems possible by one of such methods to definitely let apharmaceutical agent act in the central nervous system, they have aproblem as being highly invasive.

There have been reported various methods to let a macromolecularsubstance get into the brain through the blood-brain barrier, in whichthe macromolecular substance is modified to give it an affinity tomembrane proteins occurring on the endothelial cells of the braincapillaries. Examples of those membrane proteins which occur on theendothelial cells of the brain capillaries include receptors forcompounds such as insulin, transferrin, insulin-like growth factor(IGF-I, IGF-II), LDL, and leptin.

For example, a technique has been reported in which nerve growth factor(NGF) was synthesized into the form of a fusion protein with insulin,and this fusion protein was allowed to pass through the blood-brainbarrier via its binding to the insulin receptor (Patent Literatures4-6). Further, a technique has been reported in which nerve growthfactor (NGF) was synthesized in the form of a fusion protein withanti-insulin receptor antibody, and this fusion protein was allowed topass through the blood-brain barrier via its binding to the insulinreceptor (Patent Literatures 4 and 7). Further, a technique has beenreported in which nerve growth factor (NGF) was synthesized in the formof a fusion protein with transferrin, and this fusion protein wasallowed to pass through the blood-brain barrier via its binding to thetransferrin receptor (TfR) (Patent Literature 8). Further, a techniquehas been reported in which nerve growth factor (NGF) was synthesized inthe form of a fusion protein with anti-transferrin receptor antibody(anti-TfR antibody), and this fusion protein is allowed to pass throughthe blood-brain barrier via its binding to TfR (Patent Literatures 4 and9).

Looking further into the techniques that utilize an anti-transferrinreceptor antibody, there has been reported that in the field of thetechnique to make a pharmaceutical agent pass through the blood-brainbarrier by binding it to an anti-TfR antibody, a single-chain antibodycould be used (Non Patent Literature 3). Further, it has been reportedthat anti-hTfR antibodies exhibiting relatively high dissociationconstants with hTfR (low-affinity anti-hTfR antibody) could be favorablyused in the technique to make pharmaceutical agents pass through theblood-brain barrier (Patent Literatures 10 and 11, and Non PatentLiterature 4). Still further, it has also been reported that an anti-TfRantibodies whose affinity to hTfR varies depending on pH could beemployed as a carrier for making pharmaceutical agents pass through theblood-brain barrier (Patent Literature 12, and Non Patent Literature 5).

CITATION LIST Patent Literature

Patent Literature 1: JP2007-504166 A1

Patent Literature 2: JP2009-525963 A1

Patent Literature 3: JP2012-62312 A1

Patent Literature 4: U.S. Pat. No. 5,154,924 B1

Patent Literature 5: JP2011-144178 A1

Patent Literature 6: US2004/0101904 A1

Patent Literature 7: JP2006-511516 A1

Patent Literature 8: JPH06-228199 A1

Patent Literature 9: U.S. Pat. No. 5,977,307 B1

Patent Literature 10: WO 2012/075037

Patent Literature 11: WO 2013/177062

Patent Literature 12: WO 2012/143379

Non Patent Literature

Non Patent Literature 1: Xie Y. et al., J Control Release. 105. 106-19(2005)

Non Patent Literature 2: Ou L. et al., Mol Genet Metab. 111. 116-22(2014)

Non Patent Literature 3: Li J Y. Protein Engineering. 12. 787-96 (1999)

Non Patent Literature 4: Bien-Ly N. et al., J Exp Med. 211. 233-44(2014)

Non Patent Literature 5: Sada H. PLoS ONE. 9. E96340 (2014)

SUMMARY OF INVENTION Problems to be Solved by the Invention

Against the above background, it is an objective of the presentinvention to provide an anti-TfR antibody that can be utilized forconjugation with a compound that needs to exhibit its function in thecentral nervous system when administered parenterally (a protein or alow-molecular-weight compound and the like) in order to make thatcompound able to pass through the blood-brain barrier, and also a methodfor production thereof, as well as a method of use thereof.

Means for Solving the Problems

As a result of intense studies aimed at the above objective, the presentinventors have found that anti-human transferrin receptor antibodies(anti-hTfR antibodies) that recognize the extracellular region of hTfRwhich are obtained by the method for antibody production described indetail in the specification, efficiently passes through the blood-brainbarrier when administered to the body, and have completed the presentinvention thereupon. Thus the present invention provides what follows:

1. An anti-human transferrin receptor antibody, wherein in the heavychain variable region of the antibody,

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 orSEQ ID NO: 63,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 orSEQ ID NO: 14, and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 orSEQ ID NO: 16.

2. The antibody according to 1 above, wherein the framework region 3 ofthe heavy chain comprises the amino acid sequence set forth as SEQ IDNO: 64.

3. The antibody according to 1 or 2 above, wherein in the heavy chainvariable region,

the CDR2 comprises an amino acid sequence having a homology not lowerthan 80% to the amino acid sequence set forth as SEQ ID NO: 13 or SEQ IDNO: 14, in place thereof, and

the CDR3 comprises an amino acid sequence having a homology not lowerthan 80% to the amino acid sequence set forth as SEQ ID NO: 15 or SEQ IDNO: 16, in place thereof.

4. The antibody according to 1 or 2 above, wherein in the heavy chainvariable region,

the CDR2 comprises an amino acid sequence having a homology not lowerthan 90% to the amino acid sequence set forth as SEQ ID NO: 13 or SEQ IDNO: 14, in place thereof, and

the CDR3 comprises an amino acid sequence having a homology not lowerthan 90% to the amino acid sequence set forth as SEQ ID NO: 15 or SEQ IDNO: 16, in place thereof.

5. The antibody according to 1 or 2 above, wherein the heavy chainvariable region comprises an amino acid sequence modified from at leastone amino acid sequence of

(a) SEQ ID NO: 62 or SEQ ID NO: 63 as to the CDR1,

(b) SEQ ID NO: 13 or SEQ ID NO: 14 as to the CDR2,

(c) SEQ ID NO: 15 or SEQ ID NO: 16 as to the CDR3, and

(d) SEQ ID NO: 64 as to the framework region 3,

by the substitution, deletion or addition of 1 to 5 amino acids, inplace thereof,

wherein in the CDR1, methionine positioned at position 5 from theN-terminal side of the amino acid sequence set forth as SEQ ID NO: 62 orSEQ ID NO: 63 is also located at the same position in the amino acidsequence thus modified, and

in the framework region 3, leucine positioned at position 17 from theN-terminal side of SEQ ID NO: 64 is also located at the same position inthe amino acid sequence thus modified.

6. The antibody according to 1 or 2 above, wherein the heavy chainvariable region comprises an amino acid sequence modified from at leastone amino acid sequence of

(a) SEQ ID NO: 62 or SEQ ID NO: 63 as to the CDR1,

(b) SEQ ID NO: 13 or SEQ ID NO: 14 as to the CDR2,

(c) SEQ ID NO: 15 or SEQ ID NO: 16 as to the CDR3, and

(d) SEQ ID NO: 64 as to the framework region 3,

by the substitution, deletion or addition of 1 to 3 amino acids, inplace thereof,

wherein in the CDR1, methionine positioned at position 5 from theN-terminal side of the amino acid sequence set forth as SEQ ID NO: 62 orSEQ ID NO: 63 is also located at the same position in the amino acidsequence thus modified, and

in the framework region 3, leucine positioned at position 17 from theN-terminal side of SEQ ID NO: 64 is also located at the same position inthe amino acid sequence thus modified.

7. The antibody according to 2 above, wherein the heavy chain variableregion comprises the amino acid sequence set forth as SEQ ID NO: 65.

8. The antibody according to 7 above, wherein the heavy chain variableregion comprises, in a portion except for the amino acid sequences setforth as SEQ ID NO: 62 or SEQ ID NO: 63 of the CDR1 and SEQ ID NO: 64 ofthe framework region 3, an amino acid sequence having a homology notlower than 80% to the portion in place of the portion.

9. The antibody according to 7 above, wherein the heavy chain variableregion comprises, in a portion except for the amino acid sequences setforth as SEQ ID NO: 62 or SEQ ID NO: 63 of the CDR1 and SEQ ID NO: 64 ofthe framework region 3, an amino acid sequence having a homology notlower than 90% to the portion in place of the portion.

10. The antibody according to 7 above, wherein the antibody comprises anamino acid sequence modified from the amino acid sequence constitutingthe heavy chain variable region, by the substitution, deletion oraddition of 1 to 5 amino acids, in place thereof,

wherein in the CDR1, methionine positioned at position 5 from theN-terminus of the amino acid sequence set forth as SEQ ID NO: 63 is alsolocated at the same position in the amino acid sequence thus modified,and in the framework region 3, leucine positioned at position 17 fromthe N-terminal side of SEQ ID NO: 64 is also located at the sameposition in the amino acid sequence thus modified.

11. The antibody according to 7 above, wherein the antibody comprises anamino acid sequence modified from the amino acid sequence constitutingthe heavy chain variable region, by the substitution, deletion oraddition of 1 to 3 amino acids, in place thereof,

wherein in the CDR1, methionine positioned at position 5 from theN-terminus of the amino acid sequence set forth as SEQ ID NO: 63 is alsolocated at the same position in the amino acid sequence thus modified,and in the framework region 3, leucine positioned at position 17 fromthe N-terminal side of SEQ ID NO: 64 is also located at the sameposition in the amino acid sequence thus modified.

12. The antibody according to 7 above, wherein the heavy chain comprisesthe amino acid sequence set forth as SEQ ID NO: 66 or SEQ ID NO: 68.

13. The antibody according to 12 above, wherein the heavy chaincomprises an amino acid sequence having a homology not lower than 80% toa portion except for the amino acid sequences set forth as SEQ ID NO: 62or SEQ ID NO: 63 of the CDR1 and SEQ ID NO: 64 of the framework region3, in place of the portion.

14. The antibody according to 12 above, wherein the heavy chaincomprises an amino acid sequence having a homology not lower than 90% toa portion except for the amino acid sequences set forth as SEQ ID NO: 62or SEQ ID NO: 63 of the CDR1 and SEQ ID NO: 64 of the framework region3, in place of the portion.

15. The antibody according to 12 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequenceconstituting the heavy chain, by the substitution, deletion or additionof 1 to 5 amino acids, in place thereof, wherein in the CDR1, methioninepositioned at position 5 from the N-terminus of the amino acid sequenceset forth as SEQ ID NO: 63 is also located at the same position in theamino acid sequence thus modified, and in the framework region 3,leucine positioned at position 17 from the N-terminal side of SEQ ID NO:64 is also located at the same position in the amino acid sequence thusmodified.

16. The antibody according to 12 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequenceconstituting the heavy chain, by the substitution, deletion or additionof 1 to 3 amino acids, in place thereof, wherein in the CDR1, methioninepositioned at position 5 from the N-terminus of the amino acid sequenceset forth as SEQ ID NO: 63 is also located at the same position in theamino acid sequence thus modified, and in the framework region 3,leucine positioned at position 17 from the N-terminal side of SEQ ID NO:64 is also located at the same position in the amino acid sequence thusmodified.

17. The antibody according to one of 1 to 16 above, wherein in the lightchain variable region of the antibody,

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 6 orSEQ ID NO: 7,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 8 orSEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 10.

18. The antibody according to 17 above, wherein in the light chainvariable region,

(a) the CDR1 comprises an amino acid sequence having a homology notlower than 80% to the amino acid sequence set forth as SEQ ID NO: 6 orSEQ ID NO: 7, in place thereof,

(b) the CDR2 comprises an amino acid sequence having a homology notlower than 80% to the amino acid sequence set forth as SEQ ID NO: 8 orSEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, in place thereof,and

(c) the CDR3 comprises an amino acid sequence having a homology notlower than 80% to the amino acid sequence set forth as SEQ ID NO: 10, inplace thereof.

19. The antibody according to 17 above, wherein in the light chainvariable region,

(a) the CDR1 comprises an amino acid sequence having a homology notlower than 90% to the amino acid sequence set forth as SEQ ID NO: 6 orSEQ ID NO: 7, in place thereof,

(b) the CDR2 comprises an amino acid sequence having a homology notlower than 90% to the amino acid sequence set forth as SEQ ID NO: 8 orSEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, in place thereof,and

(c) the CDR3 comprises an amino acid sequence having a homology notlower than 90% to the amino acid sequence set forth as SEQ ID NO: 10, inplace thereof.

20. The antibody according to 17 above, wherein the light chain variableregion comprises an amino acid sequence modified from at least one aminoacid sequence of

(a) SEQ ID NO: 6 or SEQ ID NO: 7 as to the CDR1,

(b) SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Seras to the CDR2, and

(c) SEQ ID NO: 10 as to the CDR3,

by the substitution, deletion or addition of 1 to 5 amino acids, inplace thereof.

21. The antibody according to 17 above, wherein the light chain variableregion comprises an amino acid sequence modified from at least one aminoacid sequence of

(a) SEQ ID NO: 6 or SEQ ID NO: 7 as to the CDR1,

(b) SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Seras to the CDR2, and

(c) SEQ ID NO: 10 as to the CDR3,

by the substitution, deletion or addition of 1 to 3 amino acids, inplace thereof.

22. The antibody according to one of 1 to 16 above, wherein the lightchain variable region of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21 or SEQ ID NO: 22.

23. The antibody according to 22 above, wherein the antibody comprisesan amino acid sequence having a homology not lower than 80% to the aminoacid sequence of the light chain variable region, in place thereof.

24. The antibody according to 22 above, wherein the antibody comprisesan amino acid sequence having a homology not lower than 90% to the aminoacid sequence of the light chain variable region, in place thereof.

25. The antibody according to 22 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequence of thelight chain variable region by the substitution, deletion or addition of1 to 5 amino acids, in place thereof.

26. The antibody according to 22 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequence of thelight chain variable region by the substitution, deletion or addition of1 to 3 amino acids, in place thereof.

27. The antibody according to one of 1 to 16 above, wherein the lightchain of the antibody comprises the amino acid sequence set forth as SEQID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29.

28. The antibody according to 27 above, wherein the antibody comprisesan amino acid sequence having a homology not lower than 80% to the aminoacid sequence of the light chain, in place thereof.

29. The antibody according to 27 above, wherein the antibody comprisesan amino acid sequence having a homology not lower than 90% to the aminoacid sequence of the light chain, in place thereof.

30. The antibody according to 27 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequence of thelight chain by the substitution, deletion or addition of 1 to 5 aminoacids, in place thereof.

31. The antibody according to 27 above, wherein the antibody comprisesan amino acid sequence modified from the amino acid sequence of thelight chain by the substitution, deletion or addition of 1 to 3 aminoacids, in place thereof.

32. The antibody according to one of 1 to 31 above, wherein the antibodyhas an affinity to both the extracellular region of human transferrinreceptor and the extracellular region of monkey transferrin receptor.

33. The antibody according to 32 above, wherein the dissociationconstant of its complex with the extracellular region of humantransferrin receptor is not greater than 1×10⁻¹⁰ M, and the dissociationconstant of its complex with the extracellular region of monkeytransferrin receptor is not greater than 1×10⁻⁹ M.

34. The antibody according to one of 1 to 33 above, wherein the antibodyis Fab antibody, F(ab′)₂ antibody, or F(ab′) antibody.

35. The anti-human transferrin receptor antibody according to one of 1to 33 above, wherein the antibody is a single-chain antibody selectedfrom the group consisting of scFab, scF(ab′), scF(ab′)₂ and scFv.

36. The antibody according to 35 above, wherein the light chain and theheavy chain thereof are linked via a linker sequence.

37. The antibody according to 36 above, wherein the heavy chain islinked, via a linker sequence, to the light chain on the C-terminal sidethereof.

38. The antibody according to 35 above, wherein the light chain islinked, via a linker sequence, to the heavy chain on the C-terminal sidethereof.

39. The antibody according to one of 36 to 38 above, wherein the linkersequence consists of 8 to 50 amino acid residues.

40. The antibody according to 39 above, wherein the linker sequence isselected from the group consisting of the amino acid sequence Gly-Ser,the amino acid sequence Gly-Gly-Ser, the amino acid sequenceGly-Gly-Gly, the amino acid sequences set forth as SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, the amino acid sequence consisting of threeconsecutively linked amino acid sequences each set forth as SEQ ID NO:3, and the amino acid sequences consisting of 1 to 10 thereof that areconsecutively linked.

41. A fusion protein of an anti-human transferrin receptor antibody anda different protein (A), wherein

the anti-human transferrin receptor antibody is the antibody accordingto one of 1 to 40 above, and

the protein (A) is linked to the light chain of the antibody on theC-terminal side or the N-terminal side thereof.

42. The fusion protein according to 41 above, wherein the protein (A) islinked, directly or via a linker, to the light chain on the C-terminalside or the N-terminal side thereof.

43. The fusion protein according to 41 or 42 above, wherein the protein(A) is linked, via a linker, to the light chain on the C-terminal sideor the N-terminal side thereof.

44. The fusion protein according to 43 above, wherein the linker is apeptide consisting of 1 to 50 amino acid residues.

45. The fusion protein according to 44 above, wherein the linker is apeptide comprising an amino acid sequence selected from the groupconsisting of a single glycine, a single serine, the amino acid sequenceGly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequenceset forth as SEQ ID NO: 3, the amino acid sequence set forth as SEQ IDNO: 4, the amino acid sequence set forth as SEQ ID NO: 5, and the aminoacid sequences consisting of 1 to 10 thereof that are consecutivelylinked.

46. A fusion protein of an anti-human transferrin receptor antibody anda different protein (A), wherein

the anti-human transferrin receptor antibody is the antibody accordingto one of 1 to 40 above, and

the protein (A) is linked to the heavy chain of the antibody on theC-terminal side or the N-terminal side thereof.

47. The fusion protein according to 46 above, wherein the protein (A) islinked, directly or via a linker, to the heavy chain on the C-terminalside or the N-terminal side thereof.

48. The fusion protein according to 46 or 47 above, wherein the protein(A) is linked, via a linker, to the heavy chain on the C-terminal sideor the N-terminal side thereof.

49. The fusion protein according to 48 above, wherein the linkersequence is a peptide consisting of 1 to 50 amino acid residues.

50. The fusion protein according to 49 above, wherein the linker is apeptide comprising an amino acid sequence selected from the groupconsisting of a single glycine, a single serine, the amino acid sequenceGly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequenceset forth as SEQ ID NO: 3, the amino acid sequence set forth as SEQ IDNO: 4, the amino acid sequence set forth as SEQ ID NO: 5, and the aminoacid sequences consisting of 1 to 10 thereof that are consecutivelylinked.

51. The fusion protein according to one of 41 to 50 above, wherein theprotein (A) is a protein originating from human.

52. The fusion protein according to one of 41 to 51 above, wherein theprotein (A) is selected from the group consisting of nerve growth factor(NGF), lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cellline-derived neurotrophic factor (GDNF), neurotrophin-3,neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin,darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblastgrowth factor 2 (FGF2), epidermal growth factor (EGF), vascularendothelial growth factor (VEGF), interferon α, interferon β, interferonγ, interleukin 6, granulocyte-macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), cytokines, tumor necrosis factor αreceptor (TNF-α receptor), PD-1 ligands, PD-L1, PD-L2, enzymes havingβ-amyloid-degrading activity, anti-β-amyloid antibody, anti-BACEantibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody,anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody,anti-CTLA-4 antibody, and other antibody medicines.

53. A fusion protein, wherein the protein (A) is a lysosomal enzyme,wherein the lysosomal enzyme is selected from the group consisting ofα-L-iduronidase, iduronate 2-sulfatase, human acidic α-glucosidase,glucocerebrosidase, β-galactosidase, GM2 activator protein,β-hexosaminidase A, β-hexosaminidase B,N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase,galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase,aspartylglucosaminidase, α-N-acetylgalactosaminidase, acidicsphingomyelinase, α-galactosidase A, β-glucuronidase, heparanN-sulfatase, α-N-acetylglucosaminidase, acetyl CoA:α-glucosaminideN-acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, acidceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl proteinthioesterase 1, tripeptidyl-peptidase 1, hyaluronidase 1, CLN1 and CLN2.

54. The fusion protein according to one of 41 to 51 above, wherein theprotein (A) is human iduronate 2-sulfatase, human acidic α-glucosidase,or human α-L-iduronidase.

55. The fusion protein according to 51 above, wherein the protein (A) ishuman acidic α-glucosidase, wherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody is linked, on the C-terminal sidethereof and via the amino acid sequence Gly-Ser, to the human acidicα-glucosidase, thereby forming the amino acid sequence set forth as SEQID NO: 57 or SEQ ID NO: 58.

56. The fusion protein according to 51 above, wherein the protein (A) ishuman acidic α-glucosidase, wherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 66 or SEQ ID NO: 68, and the heavy chain islinked, on the C-terminal side thereof and via the amino acid sequenceGly-Ser, to the human acidic α-glucosidase having the amino acidsequence set forth as SEQ ID NO: 55 or 56.

57. The fusion protein according to 51 above, wherein the protein (A) ishuman acidic α-glucosidase, wherein the antibody is Fab antibody, andwherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody is linked, on the C-terminal sidethereof and via the amino acid sequence consisting of threeconsecutively linked amino acid sequences each set forth as SEQ ID NO:3, to the human acidic α-glucosidase, thereby forming the amino acidsequence set forth as SEQ ID NO: 89.

58. The fusion protein according to 51 above, wherein the protein (A) ishuman acidic α-glucosidase, wherein the antibody is Fab antibody, andwherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 61, and the heavy chain is linked, on theC-terminal side thereof and via the amino acid sequence consisting ofthree consecutively linked amino acid sequences each set forth as SEQ IDNO: 3, to the human acidic α-glucosidase having the amino acid sequenceset forth as SEQ ID NO: 55 or 56.

59. The fusion protein according to 51 above, wherein the protein (A) ishuman α-L-iduronidase, wherein the antibody is Fab antibody, and wherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody is linked, on the C-terminal sidethereof and via the amino acid sequence consisting of threeconsecutively linked amino acid sequences each set forth as SEQ ID NO:3, to the human α-L-iduronidase, thereby forming the amino acid sequenceset forth as SEQ ID NO: 93.

60. The fusion protein according to 51 above, wherein the protein (A) ishuman α-L-iduronidase, wherein the antibody is Fab antibody, and wherein

(1) the light chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 23, and

(2) the heavy chain of the antibody comprises the amino acid sequenceset forth as SEQ ID NO: 61, and the heavy chain is linked, on theC-terminal side thereof and via the amino acid sequence consisting ofthree consecutively linked amino acid sequences each set forth as SEQ IDNO: 3, to the human α-L-iduronidase having the amino acid sequence setforth as SEQ ID NO: 75 or 76.

61. The fusion protein according to 41 above, wherein a human IgG Fcregion or part thereof is introduced between the protein (A) and theantibody.

62. The fusion protein according to 61 above, wherein the human IgG Fcregion is linked, directly or via a linker sequence, to the protein (A)on the C-terminal side thereof, and the heavy chain or the light chainof the antibody is linked, directly or via a linker sequence, to thehuman IgG Fc region on the C-terminal side thereof.

63. The fusion protein according to 61 or 62 above, wherein the humanIgG Fc region comprises the amino acid sequence set forth as SEQ ID NO:70.

64. The fusion protein according to 63 above, wherein the human IgG Fcregion is linked, via a linker sequence, to a Fab heavy chain consistingof the amino acid sequence set forth as SEQ ID NO: 61, and comprises theamino acid sequence set forth as SEQ ID NO: 71 formed thereby.

65. A DNA fragment encoding the amino acid sequence of the anti-humantransferrin receptor antibody according to one of 1 to 40 above.

66. A DNA fragment encoding the amino acid sequence of the fusionprotein according to one of 41 to 64 above.

67. An expression vector comprising the DNA fragment according to 65 or66 above that is incorporated therein.

68. A mammalian cell transformed with the expression vector according to67 above.

69. An anti-human transferrin receptor antibody-pharmacologically activecompound complex, wherein the light chain and/or the heavy chain of theanti-human transferrin receptor antibody according to one of 1 to 40above is linked to a low-molecular-weight pharmacologically activecompound that needs to be allowed to pass through the blood-brainbarrier and exhibit the function thereof in the brain.

70. The anti-human transferrin receptor antibody-pharmacologicallyactive compound complex according to 69 above, wherein thepharmacologically active compound is any one selected from the groupconsisting of anticancer drug, therapeutic agent for Alzheimer'sdisease, therapeutic agent for Parkinson's disease, therapeutic agentfor Huntington's disease, therapeutic agent for schizophrenia,antidepressant, therapeutic agent for multiple sclerosis, therapeuticagent for amyotrophic lateral sclerosis, therapeutic agent for tumors ofcentral nervous system including brain tumor, therapeutic agent forlysosomal storage disease accompanied by encephalopathy, therapeuticagent for glycogenosis, therapeutic agent for muscular dystrophy,therapeutic agent for cerebral ischemia, therapeutic agent for priondiseases, therapeutic agent for traumatic central nervous systemdisorders, therapeutic agent for viral and bacterial central nervoussystem diseases, pharmaceutical agent used for recovery after brainsurgery, pharmaceutical agent used for recovery after spinal surgery,siRNA, antisense DNA, and peptide.

71. Use of the anti-human transferrin receptor antibody according to oneof 1 to 40 above for allowing the protein (A) or a low-molecular-weightpharmacologically active compound to pass through the blood-brainbarrier and exhibit the function thereof in the brain.

72. Use of the anti-human transferrin receptor antibody according to oneof 1 to 40 above for the production of a pharmaceutical agent forparenteral administration for the treatment of a disease condition ofthe central nervous system, by linking thereto the molecule of aphysiologically active protein or a pharmacologically activelow-molecular-weight compound for the disease condition.

73. A method for treatment of a disease condition of the central nervoussystem comprising parenterally administering to a patient with thedisorder a therapeutically effective amount of the physiologicallyactive protein, or pharmacologically active low-molecular-weightcompound, for the disorder, in the form of a conjugate with the moleculeof the anti-human transferrin receptor antibody according to one of 1 to40 above.

74. Use of the fusion protein according to one of 52 to 58 above formaking human acidic α-glucosidase pass through the blood-brain barrierand exhibit the function thereof in the brain.

75. Use of the fusion protein according to one of 52 to 58 above for theproduction of a pharmaceutical agent for parenteral administration forthe treatment of a disease condition of the central nervous systemaccompanying Pompe's disease.

76. A method for the treatment of a disease of the central nervoussystem accompanying Pompe's disease comprising parenterallyadministering a therapeutically effective amount of the fusion proteinaccording to one of 52 to 58 above to a patient with the disease.

77. Use of the fusion protein according to one of 52 to 54, 59, and 60above for making human acidic α-L-iduronidase pass through theblood-brain barrier and exhibit the function thereof in the brain.

78. Use of the fusion protein according to one of 52 to 54, 59, and 60above for the production of a pharmaceutical agent for parenteraladministration for the treatment of a disease condition of the centralnervous system accompanying Hurler syndrome or Hurler-Scheie syndrome.

79. A method for the treatment of a disease of the central nervoussystem accompanying Hurler syndrome or Hurler-Scheie syndrome, themethod comprising parenterally administering a therapeutically effectiveamount of the fusion protein according to one of 52 to 54, 59, and 60above to a patient with the disease.

Effects of the Invention

By the present invention, various compounds, such as proteins andlow-molecular-weight compounds that, although physiologically orpharmacologically active, have been unusable by parenteraladministration because of their no or little ability to pass through theblood-brain barrier, can be provided in the form that allow them to passthrough the blood-brain barrier, thus making them new pharmaceuticalagents for parenteral administration for the treatment of a diseasecondition of the central nervous system.

BRIEF DESCRIPTION OF DRAWINGS

[FIG. 1] Substitute photographs for drawings showing the result of theimmunohistochemical staining of the anti-hTfR antibody in the cerebralcortex of a crab-eating monkey after a single intravenous administrationof the anti-hTfR antibody. (a) anti-hTfR antibody not administered, (b)anti-hTfR antibody No.3 administered. The bar at the bottom right ineach photograph is a 50-μm gauge.

[FIG. 2] A figure showing the result of the immunohistochemical stainingof the anti-hTfR antibody in the hippocampus of a crab-eating monkeyafter a single intravenous administration of the anti-hTfR antibody. (a)anti-hTfR antibody not administered, (b) anti-hTfR antibody No.3administered. The bar at the bottom right in each photograph is a 50-μmgauge.

[FIG. 3] Substitute photographs for drawings showing the result of theimmunohistochemical staining of the anti-hTfR antibody in the cerebellumof a crab-eating monkey after a single intravenous administration of theanti-hTfR antibody. (a) anti-hTfR antibody not administered, (b)anti-hTfR antibody No.3 administered. The bar at the bottom right ineach photograph is a 50-μm gauge.

[FIG. 4] A figure showing the amount of a humanized anti-hTfR antibodyaccumulated in various organs other than the brain of a crab-eatingmonkey after a single intravenous administration. The vertical axisindicates the amount of the humanized anti-hTfR antibody (μg/g wetweight) per wet weight of each organ. The white bars represent, from theleft, the amount accumulated in each organ of the monkey afteradministration of humanized anti-hTfR antibody No.3, humanized anti-hTfRantibody No.3-2, humanized anti-hTfR antibody No.3 (IgG4), and humanizedanti-hTfR antibody No.3-2 (IgG4), respectively, and the black barsrepresent the amount accumulated in respective organs of the monkeyafter administration of trastuzumab (Herceptin™). “ND” denotes “notdetected”.

[FIG. 5] Substitute photographs for drawings showing the result ofimmunohistochemical staining of a humanized anti-hTfR antibody in thecerebral cortex of a crab-eating monkey after a single intravenousadministration. (a) Herceptin administered, (b) humanized anti-hTfRantibody No.3 administered, (c) humanized anti-hTfR antibody No.3-2administered, (d) humanized anti-hTfR antibody No.3 (IgG4) administered,(e) humanized anti-hTfR antibody No.3-2 (IgG4) administered. The bar atthe bottom right in each photograph is a 20-μm gauge.

[FIG. 6] Substitute photographs for drawing showing the result ofimmunohistochemical staining of a humanized anti-hTfR antibody in thehippocampus of a crab-eating monkey after a single intravenousadministration. (a) Herceptin administered, (b) humanized anti-hTfRantibody No.3 administered, (c) humanized anti-hTfR antibody No.3-2administered, (d) humanized anti-hTfR antibody No.3 (IgG4) administered,(e) humanized anti-hTfR antibody No.3-2 (IgG4) administered. The bar atthe bottom right at each photograph is a 20-μm gauge.

[FIG. 7] A figure showing the result of immunohistochemical staining ofhumanized anti-hTfR antibody in the cerebellum of a crab-eating monkeyafter a single intravenous administration. (a) Herceptin administered,(b) humanized anti-hTfR antibody No.3 administered, (c) humanizedanti-hTfR antibody No.3-2 administered, (d) humanized anti-hTfR antibodyNo.3 (IgG4) administered, (e) humanized anti-hTfR antibody No.3-2 (IgG4)administered. The bar at the bottom right at each photograph is a 20-μmgauge.

[FIG. 8] A figure showing the result of immunohistochemical staining ofhumanized anti-hTfR antibody in the medulla oblongata of a crab-eatingmonkey after a single intravenous administration. (a) Herceptinadministered, (b) humanized anti-hTfR antibody No.3 administered, (c)humanized anti-hTfR antibody No.3-2 administered, (d) humanizedanti-hTfR antibody No.3 (IgG4) administered, (e) humanized anti-hTfRantibody No.3-2 (IgG4) administered. The bar at the bottom right at eachphotograph is a 20-μm gauge.

[FIG. 9] A figure showing the result of immunohistochemical staining ofhGAA in the cerebellum of a crab-eating monkey after a singleintravenous administration. (a) hGAA-anti-hTfR antibody 3N (IgG4)administered, (b) hGAA administered. The bar at the bottom right at eachphotograph is a 20-μm gauge.

[FIG. 10] A figure showing the result of assessment of thepharmacological effect of hGAA-anti-hTfR antibody 3N (IgG4) using amouse. The concentrations of glycogen in (a) right brain, (b) cervicalpart of spinal cord, (c) heart, (d) diaphragm, (e) liver and (f) spleenare given. In each figure, numeral 1 indicates a normal control group,numeral 2 indicates a disease control group, numeral 3 indicates a groupadministered with 20 mg/kg of hGAA, and numerals 4 to 7 indicate groupsadministered with 2.5 mg/kg, 5.0 mg/kg, 10 mg/kg and 20 mg/kg,respectively, of hGAA-anti-hTfR antibody 3N (IgG4). The ordinateindicates the concentration of glycogen (mg/g in terms of a wet weight).The vertical bar indicates SD, “#” indicates p<0.01 compared with thedisease control group, “##” indicates p<0.05 compared with the diseasecontrol group, “$” indicates p<0.01 compared with the hGAA administeredgroup, and “$$” indicates p<0.05 compared with the hGAA administeredgroup (each based on Turkey HSD test).

[FIG. 11] A figure showing the result of assessment of thepharmacological effect of hGAA-anti-hTfR antibody 3N (IgG4) using amouse. The concentrations of glycogen in (a) quadriceps femoris muscle,(b) gastrocnemius muscle, (c) soleus muscle, (d) tibialis anteriormuscle and (e) extensor digitorum longus muscle gastrocnemius. In eachfigure, numeral 1 indicates a normal control group, numeral 2 indicatesa disease control group, numeral 3 indicates a hGAA administered group,and numerals 4 to 7 indicate groups administered with 2.5 mg/kg, 5.0mg/kg, 10 mg/kg and 20 mg/kg, respectively, of hGAA-anti-hTfR antibody3N (IgG4). The ordinate indicates the concentration of glycogen (mg/g interms of a wet weight). The vertical bar indicates SD, “#” indicatesp<0.01 compared with the disease control group, “##” indicates p<0.05compared with the disease control group, “$” indicates p<0.01 comparedwith the hGAA administered group, and “$$” indicates p<0.05 comparedwith the hGAA administered group (each based on Turkey HSD test).

[FIG. 12] A figure showing the result of assessment of thepharmacological effect of Fab GS-GAA using a mouse. The concentrationsof glycogen in (a) right brain, (b) heart, (c) diaphragm, (d) quadricepsfemoris muscle, (e) soleus muscle and (f) tibialis anterior muscle. Ineach figure, numeral 1 indicates a normal control group, numeral 2indicates a disease control group, numeral 3 indicates a hGAAadministered group, and numerals 4 and 5 indicate 5.0 mg/kg and 20mg/kg, respectively, of Fab GS-GAA. The ordinate indicates theconcentration of glycogen (mg/g in terms of a wet weight). The verticalbar indicates SD.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

In the present invention, the term “antibody” refers mainly to a humanantibody, mouse antibody, humanized antibody, as well as a chimericantibody between human antibody and non-human mammalian antibody, and achimeric antibody between mouse antibody and non-mouse mammalianantibody, but the meaning of the term is not limited to them insofar asa substance of interest has a property to specifically bind to a certainantigen, and there is no specific limitation as to the animal species ofthe antibody, either. However, preferred is a humanized antibody.

In the present invention, the term “human antibody” refers to anantibody whose entire protein is encoded by a gene originating fromhuman. The term “human antibody”, however, also includes an antibodyencoded by a gene obtained by introducing a mutation into an originalhuman gene for a purpose of enhancing expression efficacy of the gene,for example, without modifying the original amino acid sequence. Theterm “human antibody” also includes an antibody which is produced bycombining two or more genes encoding human antibodies and replacingcertain part of a human antibody with part of another human antibody. Ahuman antibody includes three complementarity determining regions (CDRs)in the light chain of the immunoglobulin and three complementaritydetermining regions (CDRs) in the heavy chain of the immunoglobulin. Thethree CDRs in the light chain of the immunoglobulin are called, from theN-terminal side, CDR1, CDR2 and CDR3, respectively. The three CDRs inthe heavy chain of the immunoglobulin are also called, from theN-terminal side, CDR1, CDR2 and CDR3, respectively. The term “humanantibody” also includes a human antibody produced by replacing a CDR ofa human antibody with a CDR of another human antibody to modify suchproperties as the antigen specificity and the affinity of the originalhuman antibodies, etc.

In the present invention, the term “human antibody” also includes anantibody which is produced through modification of the gene of theoriginal human antibody by introducing a mutation, such as substitution,deletion, addition, to the amino acid sequence of the original antibody.When replacing one or more amino acids of the amino acid sequence of theoriginal antibody with other amino acids, the number of amino acidreplaced may preferably be 1-20, more preferably 1-5, and still morepreferably 1-3. When deleting one or more amino acids of the amino acidsequence of the original antibody, the number of amino acids deleted maypreferably be 1-20, more preferably 1-5, and still more preferably 1-3.An antibody produced by a combined mutation of these substitution anddeletion of amino acids is also a “human antibody”. In some cases, oneor more amino acids, preferably 1-20, more preferably 1-5, and stillmore preferably 1-3 amino acids may be added inside the amino acidsequence of the original antibody or on its N- or C-terminal side. Anantibody produced by a combined mutation of addition, substitution, anddeletion of amino acids is also a “human antibody”. The amino acidsequence of such a mutated antibody has a homology of preferably notlower than 80%, more preferably not lower than 90%, still morepreferably not lower than 95%, and even more preferably not lower than98%, to the amino acid sequence of the original antibody. Thus, in thepresent invention, the term “gene originating from human” includes notonly the unmutated gene originating from human but also a gene producedby modifying this.

The homology between the amino acid sequence of an unmutated antibodyand the amino acid sequence of an antibody produced by introducing amutation into it may be readily calculated using well-known homologycalculator algorithms As such algorithms, there are, for example, BLAST(Altschul S F. J Mol. Biol. 215. 403-10 (1990)), a similarity search byPearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), andthe local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2.482-9 (1981)), and the like.

In the present invention, the term “mouse antibody” refers to anantibody whose entire protein consists of an amino acid sequence whichis the same as an antibody encoded by a gene originating from a mouse.Therefore, the term “mouse antibody” also includes an antibody that isencoded by a gene produced by introducing a mutation into the originalmouse gene without causing a change in its amino acid sequence but inorder, for example, to improve the expression efficiency of the gene.Further, the term “mouse antibody” also includes an antibody producedthrough combining two or more genes encoding mouse antibodies byreplacing part of a mouse antibody with part of another mouse antibody.A mouse antibody has three complementarity determining regions (CDRs) inthe light chain of the immunoglobulin and three complementaritydetermining regions (CDRs) in the heavy chain of the immunoglobulin. Thethree CDRs in the light chain of the immunoglobulin are called, from theN-terminal side, CDR1, CDR2 and CDR3, respectively. The three CDRs inthe heavy chain of the immunoglobulin are also called, from theN-terminal side, CDR1, CDR2 and CDR3, respectively. The term “mouseantibody” also includes an antibody produced by replacing a CDR of amouse antibody with a CDR of another mouse antibody to modify thespecificity and affinity of the original mouse antibodies.

In the present invention, the term “mouse antibody” also includes anantibody which is produced through modification of the gene of theoriginal mouse antibody by introducing a mutation, such as substitution,deletion, addition, to the amino acid sequence of the original antibody.When replacing one or more amino acids of the amino acid sequence of theoriginal antibody with other amino acids, the number of amino acidreplaced may preferably be 1-20, more preferably 1-5, and still morepreferably 1-3. When deleting one or more amino acids of the amino acidsequence of the original antibody, the number of amino acids deleted maypreferably be 1-20, more preferably 1-5, and still more preferably 1-3.An antibody produced by a combined mutation of these substitution anddeletion of amino acids is also included in a “mouse antibody”. Whenadding one or more amino acids, they may be added inside the amino acidsequence of the original antibody or on its N- or C-terminal side,preferably 1-20, more preferably 1-5, and still more preferably 1-3, innumber. An antibody produced by a combined mutation of addition,substitution, and deletion of amino acids is also included in a “mouseantibody”. The amino acid sequence of such a mutated antibody has ahomology of preferably not lower than 80%, more preferably not lowerthan 90%, still more preferably not lower than 95%, and even morepreferably not lower than 98%, to the amino acid sequence of theoriginal antibody. Thus, in the present invention, the term “geneoriginating from mouse” includes not only the unmutated gene originatingfrom mouse but also a gene produced by modifying this.

In the present invention, the term “humanized antibody” refers to anantibody in which part of the amino acid sequence of its variable region(e.g., especially the whole or part of its CDRs) originates from anon-human mammal while the rest originates from human. An example ofhumanized antibody is an antibody produced by replacing the threecomplementarity determining regions (CDRs) of the light chain of theimmunoglobulin and the three complementarity determining regions (CDRs)of the heavy chain of the immunoglobulin constituting a human antibody,with CDRs from a non-human mammal. As far as it originates from anon-human mammal, there is no particular limitation as to the biologicalspecies from which those CDRs originate that are transplanted into aproper position of the human antibody, though preferred are mouse, rat,rabbit, horse or non-human primate, more preferred are mouse and rat,and still more preferred is mouse.

In the present invention, the term “chimeric antibody” refers to anantibody produced by connecting fragments of two or more differentantibodies originating from two or more different species.

A chimeric antibody between a human antibody and a non-human mammalianantibody is an antibody provided by replacing part of a human antibodywith part of a non-human mammalian antibody. As explained below, anantibody is made of an Fc region, a Fab region and a hinge region. Aspecific example of such chimeric antibodies is a chimeric antibodywhose Fc region originates from a human antibody while its Fab regionoriginates from a non-human mammalian antibody. The hinge region eitheroriginates from a human antibody or from a non-human mammalian antibody.On the contrary, the term chimeric antibody also includes one whose Fcregion originates from a non-human mammalian antibody while its Fabregion originates from a human antibody. In such a case also, the hingeregion either originates from a human antibody or from a non-humanmammalian antibody.

An antibody can be viewed as composed of a variable region and aconstant region. Additional examples of chimeric antibodies include anantibody in which the heavy chain constant region (C_(H)) and the lightchain constant region (C_(l)) both originate from a human antibody whilethe heavy chain variable region (V_(H)) and the light chain variableregion (V_(L)) both originate from an antibody of a non-human mammal,and conversely, an antibody in which the heavy chain constant region(C_(H)) and the light chain constant region (C_(l)) both originate froman antibody of a non-human mammal, while the heavy chain variable region(V_(H)) and the light chain variable region (V_(L)) both originate froma human antibody. In these, there is no particular limitation as to thebiological species of the non-human mammal, as far as it is a non-humanmammal, though preferred are mouse, rat, rabbit, horse or non-humanprimate, and more preferred is mouse.

A chimeric antibody between a mouse antibody and a non-mouse mammalianantibody is an antibody provided by replacing part of a mouse antibodywith part of a non-mouse mammalian antibody. Specific examples of suchchimeric antibodies include a chimeric antibody whose Fc regionoriginates from a mouse antibody while its Fab region originates from anon-mouse mammalian antibody, and conversely, a chimeric antibody whoseFc region originates from a non-mouse mammal while its Fab regionoriginates from a mouse antibody. In these, there is no particularlimitation as to the biological species of the non-mouse mammal, as faras it is a mammal other than mouse, though preferred are rat, rabbit,horse or non-human primate, and more preferred is human.

A chimeric antibody between a human antibody and a mouse antibody isdesignated in particular “human/mouse chimeric antibody”. Examples ofhuman/mouse chimeric antibodies include a chimeric antibody in which theFc region originates from a human antibody while the Fab regionoriginates from a mouse antibody, and conversely, a chimeric antibodywhose Fc region originates from mouse antibody, while its Fab regionoriginates from a human antibody. A hinge region either originates froma human antibody or a mouse antibody. Additional specific examples ofhuman/mouse chimeric antibodies include those whose heavy chain constantregion (C_(H)) and light chain constant region (C_(L)) originate from ahuman antibody while its heavy chain variable region (V_(H)) and lightchain variable region (V_(L)) originate from a mouse antibody, andconversely, those whose heavy chain constant region (C_(H)) and lightchain constant region (C_(L)) originate from a mouse antibody while itsheavy chain variable region (V_(H)) and light chain variable region(V_(L)) originate from a human antibody.

Originally, an antibody is of the basic structure having fourpolypeptide chains in total consisting of two immunoglobulin lightchains and two immunoglobulin heavy chains. However, in the presentinvention the term “antibody” refers, besides an antibody having thisbasic structure, also to:

(1) one consisting of two polypeptide chains: a single immunoglobulinlight chain and a single immunoglobulin heavy chain, and also, asexplained later,

(2) a single-chain antibody consisting of an immunoglobulin light chainwhich is linked, on the C-terminal side thereof, to a linker sequencewhich in turn is linked, on the C-terminal side thereof, to animmunoglobulin heavy chain,

(3) single-chain antibodies consisting of an immunoglobulin heavy chainwhich is linked, on the C-terminal side thereof, to a linker sequencewhich in turn is linked, on the C-terminal side thereof, to animmunoglobulin light chain, and

(4) one consisting of a Fab region, i.e., a structure left behind byremoval of the Fc region from an antibody having the basic structure, asthe original meaning, and one consisting of the Fab region and the wholeor part of the hinge region (including Fab, F(ab′), and F(ab′)₂) alsoare included in the term “antibody” in the present invention.

The term “Fab” refers to a molecule consisting of a single light chaincomprising the variable region and the C_(L) region (light chainconstant region) and a single heavy chain comprising the variable regionand the C_(H)1 region (portion 1 of heavy chain constant region) whichare combined by a disulfide bond between their respective cysteineresidues. While the heavy chain in a Fab can include part of the hingeregion in addition to the variable region and the C_(H)1 region (portion1 of heavy chain constant region), the hinge region in such a case lacksthe cysteine residue that otherwise is present in the hinge region andwould serve to link two heavy chains of an antibody together. In Fab,the light chain and the heavy chain are connected by a disulfide bondformed between the cysteine residue present in the light chain constantregion (C_(L) region) and the cysteine residue located in the heavychain constant region (C_(H)1 region) or the hinge region. The heavychain forming Fab is called a Fab heavy chain. As it lacks the cysteineresidue in the hinge region which serves to bind two heavy chains of anantibody, Fab consists of a single light chain and a single heavy chain.The light chain constituting Fab includes a variable region and a C_(L)region. The heavy chain as a component of Fab may either consist of avariable region and a C_(H)1 region or also of part of the hinge regionin addition to the variable region and the C_(H)1 region. However, inthe letter case, the hinge region is so selected as not to include thecysteine residue that could bind two heavy chains, in order to avoid theformation of a disulfide bond between two heavy chains at their hingeregions. In F(ab′), the heavy chain includes, in addition to a variableregion and a C_(H)1 region, the whole or part of a hinge regioncontaining a cysteine residue that could bind two heavy chains. F(ab′)₂is a molecule consisting of two F(ab′)s bound together through adisulfide bond formed between the cysteine residues present in theirrespective hinge regions. The heavy chain forming F(ab′) or F(ab′)₂ iscalled a Fab′ heavy chain. Further, a polymer such as a dimer and atrimer, which consists of two or more antibodies connected with eachother, directly or via a linker, is also included in the term“antibody”. Moreover, in addition to the aforementioned, any moleculethat includes part of an immunoglobulin molecule and has a property tospecifically bind to the antigen is also included in the term “antibody”in the present invention. Thus, in the present invention, the term“immunoglobulin light chain” includes a molecule that is derived from anoriginal immunoglobulin light chain and having the amino acid sequenceof the whole or part of its variable region. Likewise, the term“immunoglobulin heavy chain” includes a molecule that is derived from anoriginal immunoglobulin heavy chain and having the amino acid sequenceof the whole or part of its variable region. Therefore, insofar ashaving the whole or part of the amino acid sequence of the variableregion, a molecule is included in the term “immunoglobulin heavy chain”,even if it lacks its Fc region, for example.

In the above, the term “Fc” or “Fc region” refers to a region comprisinga fragment consisting of C_(H)2 region (portion 2 of the heavy chainconstant region), and C_(H)3 region (portion 3 of the heavy chainconstant region) in the antibody molecule.

Furthermore, in the present invention, the term “antibody” alsoincludes:

(5) scFab, scF(ab′), and scF(ab′)₂, which are single-chain antibodiesproduced by binding the light chain to the heavy chain that form,respectively, the Fab, F(ab′) and F(ab′)₂ mentioned in (4) above, via alinker sequence. Such scFab, scF(ab′) and scF(ab′)₂ may be a molecule inwhich either the light chain is linked, on the C-terminal side thereof,to a linker sequence, which in turn is linked, on the C-terminal sidethereof, to the heavy chain, or the heavy chain is linked, on theC-terminal side thereof, to a linker sequence, which in turn is linked,on the C-terminal side thereof, to the light chain. Furthermore, a scFv,which is a single-chain antibody provided by binding the light chainvariable region to the heavy chain variable region, via a linkersequence between them, is also included in the term “antibody” in thepresent invention. Such scFv may be a molecule in which either the lightchain variable region is linked, on the C-terminal side thereof, to alinker sequence, which in turn is linked, on the C-terminal sidethereof, to the heavy chain variable region, or the heavy chain variableregion is linked, on the C-terminal side thereof, to a linker sequence,which in turn is linked, on the C-terminal side thereof, to the lightchain variable region.

Furthermore, in addition to a full-length antibody and those describedin (1) to (5) above, the term “antibody” in the present specificationincludes, any form of antigen-binding fragment which lacks part of thefull-length antibody (antibody fragment), a broader concept whichincludes (4) and (5) above.

The term “antigen-binding fragment” refers to an antibody fragment thatretains at least part of the specific binding activity to its antigen.In addition to those described above in (4) and (5), examples of bindingfragments include Fab, Fab′, F(ab′)₂, variable region (Fv); asingle-chain antibody (scFv) produced by linking the heavy chainvariable region (V_(H)) and the light chain variable region (V_(L)), viaa proper linker between them; a diabody, which is a dimer of apolypeptide that comprises a heavy chain variable region (V_(H)) and alight chain variable region (V_(L)); a minibody, which is a dimer of amolecule in which the heavy chain (H chain) of a scFv is linked to partof the constant region (C_(H)3), and other low-molecular-antibodies.However, as far as it has an antigen-binding ability, the term is notlimited to these molecules. Such binding fragments include not onlythose produced by treating a full-length molecule of an antibody proteinwith a proper enzyme but also those produced by proper host cells usinga genetically engineered antibody gene.

In the present invention, the term “single-chain antibody” refers to aprotein in which an amino acid sequence comprising the whole or part ofan immunoglobulin light chain variable region linked, on the C-terminalside thereof, to a linker sequence, which in turn is linked, on theC-terminal side thereof, to the amino acid sequence of the whole or partof an immunoglobulin heavy chain variable region, and having an abilityto specifically bind a certain antigen. For example, those described in(2), (3) and (5) are included in “single-chain antibody”. Further, aprotein in which an amino acid sequence comprising the whole or part ofan immunoglobulin heavy chain variable region is linked, on theC-terminal side thereof, to a linker sequence, which in turn is furtherlinked, on the C-terminal side thereof, to the amino acid sequence ofthe whole or part of an immunoglobulin light chain variable region, andwhich has an ability to specifically bind to a certain antigen, is alsoincluded in the term “single-chain antibody” in the present invention.In a single-chain antibody in which an immunoglobulin heavy chain islinked, on the C-terminal side thereof and via a linker sequence, to animmunoglobulin light chain, the immunoglobulin heavy chain generallylacks the Fc region. An immunoglobulin light chain variable region hasthree complementarity determining regions (CDRs) which participate indetermining the antigen specificity of an antibody. Likewise, animmunoglobulin heavy chain variable region also has three CDRs. ThoseCDRs are the primary regions that determine the antigen specificity ofan antibody. Therefore, a single-chain antibody preferably contains allthe three CDRs of the immunoglobulin heavy chain and all the three CDRsof the immunoglobulin light chain. However, it is also possible toprovide a single-chain antibody in which one or more of those CDRs aredeleted, insofar as the antigen-specific affinity of the antibody isretained.

In a single-chain antibody, the linker sequence placed between the lightchain and the heavy chain of the immunoglobulin is preferably a peptidechain consisting of preferably 2 to 50, more preferably 8 to 50, stillmore preferably 10 to 30, even more preferably 12 to 18, or 15 to 25,for example 15 or 25 amino acid residues. While there is no particularlimitation as to the specific amino acid sequence of such a linkersequence insofar as the anti-hTfR antibody comprising the both chainslinked thereby retains the affinity to hTfR, it is preferably made ofglycine only, or of glycine and serine: for example the amino acidsequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acidsequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQID NO:3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4),the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequencewhich includes 2 to 10 or 2 to 5 repeats of any of those amino acidsequences. For example, in linking the amino acid sequence of the entireimmunoglobulin heavy chain variable region, on the C-terminal sidethereof and via a linker sequence, to immunoglobulin light chainvariable region, the linker sequence is preferably a linker sequencecomprising 15 amino acids corresponding to three of the amino acidsequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3) consecutively linked.

In the present invention, the term “human transferrin receptor” or“hTfR” refers to a membrane protein having the amino acid sequence setforth as SEQ ID NO:1. The anti-hTfR antibody of the present inventionis, in one of its embodiments, that which specifically binds to theamino acid sequence set forth as SEQ ID NO:1 in its part starting withthe cysteine residue at position 89 from the N-terminal side to thephenylalanine at the C-terminus (i.e., the extracellular region of thehTfR), though it is not limited to this embodiment. Further, in thepresent invention, the term “monkey transferrin receptor” or “monkeyTfR” refers in particular to the membrane protein having the amino acidsequence set forth as SEQ ID NO:2, originating from crab-eating monkey(Macaca fascicularis). The anti-hTfR antibody of the present inventionis, in one of its embodiments, that which binds also to the amino acidsequence set forth as SEQ ID NO:2 in its part starting with the cysteineresidue at position 89 from the N-terminal side to the phenylalanine atthe C-terminus (i.e., the extracellular region of the monkey TfR),though it is not limited to this embodiment.

For preparation of an antibody to hTfR, there is known a general methodaccording to which a recombinant human transferrin receptor (rhTfR) isproduced using cells which have an introduced expression vector havingan incorporated hTfR gene, and then animals such as mice are immunizedwith this rhTfR. By collecting those cells which produce antibodies tohTfR from the immunized animals and fusing them with myeloma cells,hybridoma cells can be obtained having an ability to produce theanti-hTfR antibody.

Further, cells producing an antibody to hTfR can also be obtained bycollecting immunocompetent cells from an animal such as mouse, andimmunizing them with rhTfR by in vitro immunization. In conducting invitro immunization, there is no particular limitation as to the animalspecies from which the immunocompetent cells are derived, thoughpreferred are mouse, rat, rabbit, guinea pig, dog, cat, horse, andprimates including human, and more preferred are mouse, rat and human,and still more preferably mouse and human. As mouse immunocompetentcells, spleen cells prepared from mouse spleen may be used, for example.As human immunocompetent cells, such cells can be used as prepared fromhuman peripheral blood, bone marrow, spleen, and the like. By immunizinghuman immunocompetent cells according to in vitro immunization, a humanantibody to hTfR can be obtained.

After immunizing the immunocompetent cells according to in vitroimmunization, the cells can be fused with myeloma cells to preparehybridoma cells having an ability to produce the antibody. Further, itis also possible to extract mRNAs from the immunized cells, synthesizecDNA, perform PCR reaction using the cDNA as a template to amplify a DNAfragment containing the gene encoding the light chain and the heavychain of the immunoglobulin, and artificially reconstruct the antibodygene using them.

The hybridoma cells freshly obtained above also include such cells thatproduce antibodies that recognize other proteins than hTfR. Furthermore,not all the hybridoma cells producing an anti-hTfR antibody necessarilyproduce an anti-hTfR antibody that exhibits high affinities to hTfR.

Likewise, artificially reconstructed antibody genes include such genesas encode antibodies recognizing other proteins than hTfR as antigens.Moreover, not all the genes encoding anti-hTfR antibodies necessarilyhave desired properties such as encoding an anti-hTfR antibodyexhibiting high affinity to hTfR.

Therefore, a selection step is necessary to select hybridoma cellsproducing an antibody having desired properties (such as high affinityto hTfR) from the hybridoma cells freshly obtained above. Further, inthe case where antibody genes are artificially reconstructed, aselection step is necessary to select from the antibody genes a geneencoding an antibody having desired properties (such as high affinitiesto hTfR). For selecting hybridoma cells that produce antibodiesexhibiting high affinities to hTfR (high affinity antibodies), or forselecting genes encoding high affinity antibodies, following methodsexplained in detail below are effective. Besides, antibodies exhibitinghigh affinity to hTfR are those whose dissociation constant (K_(D)) withhTfR as measured by the method described in Example 7 is preferably notgreater than 1×10⁻⁸ M, more preferably not greater than 1×10⁻⁹ M, stillmore preferably not greater than 1×10⁻¹⁰ M, and even more preferably notgreater than 1×10⁻¹¹ M. For example, those having a dissociationconstant of 1×10⁻¹³ M to 1×10⁻⁹ M, or 1×10⁻¹³ M to 1×10⁻¹⁰ M arepreferable.

For example, for selecting hybridoma cells which produce high affinityantibodies to anti-hTfR antibody, a method is employed in whichrecombinant hTfR is added to a plate and held by it, then the culturesupernatant of the hybridoma cells is added, and after removing antibodyunbound to the recombinant hTfR from the plate, the amount of theantibody held by the plate is measured. According to this method, thehigher the affinity to hTfR of the antibody contained in the culturesupernatant of the hybridoma cells added to the plate is, the greaterthe amount of antibody held by the plate becomes. Therefore, bymeasuring the amount of the antibody held by the plate, it is possibleto select those hybridoma cells corresponding to the plates where theantibody is held in the greater amount as cell lines producing ananti-hTfR antibody having the relatively higher affinity to hTfR. It isalso possible to isolate the gene encoding the high-affinity antibody byextracting mRNAs from each cell line selected in this manner,synthesizing cDNAs, and amplifying a DNA fragment containing the geneencoding the anti-hTfR antibody by PCR using the cDNA as a template.

In order to select the gene encoding the high-affinity anti-hTfRantibody from the above artificially reconstructed antibody genes, theartificially reconstructed antibody genes are once incorporated into anexpression vector, and the expression vector then is introduced intohost cells. Although there is no particular limitation as to the cellsto be employed as host cells, even whether they are prokaryoticeukaryotic, insofar as they can express the antibody gene afterintroduction of an expression vector having the incorporatedartificially reconstructed antibody gene, preferred are cellsoriginating mammals such as human, mouse, Chinese hamster, and the like,and particularly preferred are CHO cells originating from Chinesehamster ovary cells, or NS/0 cells originating from mouse myeloma.Further, there is no particular limitation as to an expression vector tobe employed for incorporation of the antibody encoding gene andexpression of it, and any expression vector may be used as far as it canexpress the gene when incorporated into mammalian cells. The geneincorporated into an expression vector is located downstream of a DNAsequence that can regulate the frequency of transcription of a gene inmammalian cells (gene expression regulatory site). Examples of geneexpression regulatory sites that may be employed in the presentinvention include cytomegalovirus-derived promoter, SV40 early promoter,human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter.

Mammalian cells having such an introduced expression vector come toexpress the artificially reconstructed antibody incorporated in theexpression vector. In order to select those cells which produce ahigh-affinity antibody to anti-hTfR antibody from the above obtainedcells expressing the artificially reconstructed antibody, a method isemployed in which the recombinant hTfR is added to a plate and held byit, then the recombinant hTfR is contacted by the culture supernatant ofthe cells, and after the removal of antibody unbound to the recombinanthTfR from the plate, the amount of the antibody held by the plate ismeasured. According to this method, the higher the affinity to hTfR ofthe antibody contained in the cells culture supernatant is, the greaterthe amount of antibody held by the plate becomes. Therefore, bymeasuring the amount of the antibody held by the plate, one can selectthose cells corresponding to the plate where the antibody is held in thegreater amount, as a cell line producing an anti-hTfR antibody havingrelatively the high-affinity anti-hTfR antibody, and eventually canselect a gene encoding an anti-hTfR antibody having a high-affinityanti-hTfR antibody to hTfR. Using cell line selected in this manner, onecan perform PCR to amplify a DNA fragment containing the gene encodingthe anti-hTfR antibody to isolate the gene encoding the high-affinityantibody.

Selection of the gene encoding a high affinity anti-hTfR antibody fromthe above artificially reconstructed antibody genes can also be carriedout by incorporating the artificially reconstructed antibody genes intoan expression vector, introducing the expression vector into E. colicells, culturing the E. coli cells, and selecting the E. coli cellshaving the desired gene, in the same manner as in the above selection ofhybridoma cells, using the culture supernatant of the E. coli cells oran antibody-containing solution prepared by lysing the E. coli cells. E.coli cells thus selected express the gene encoding an anti-hTfR antibodyhaving a relatively high affinity to hTfR. From this cell line, the geneencoding the anti-hTfR antibody having a relatively the high-affinityanti-hTfR antibody to hTfR can be selected. In order to allow theantibody to be secreted into the E. coli culture supernatant, theantibody gene may be incorporated into the expression vector so that asecretion signal sequence is attached on the N-terminal side of thegene.

Another method for selection of the gene encoding a high-affinityanti-hTfR antibody is a method in which the antibody encoded by theabove artificially reconstructed antibody gene is expressed and retainedon phage particles. For this, the antibody gene is reconstructed as agene encoding a single-chain antibody. A method for retaining phageparticles to retain an antibody on their surface is disclosed ininternational publications WO1997/09436 and WO1995/11317, and the like,and thus well known. In order to select phages retaining thehigh-affinity antibody to anti-hTfR antibody from the phages retainingthe antibodies encoded by the artificially reconstructed antibody genes,a method is employed in which a recombinant hTfR from the plate is addedto a plate and held by, contacted by the phages, and after removal ofthe phages unbound to the recombinant hTfR, the amount of the phagesheld by the plate is measured. According to this method, the higher theaffinity to hTfR of the antibody retained on the phage particles is, thegreater the amount of the phage held by the plate becomes. Therefore, bymeasuring the amount of the phage held by the plate, one can select thephage particles corresponding to the plate where the phages' were heldin the greater amount, as the phage particles producing anti-hTfRantibody having a relatively the high-affinity anti-hTfR antibody tohTfR, and eventually can select the gene encoding the high-affinityanti-hTfR antibody to hTfR. Using the phage particles thus selected, PCRcan be performed to amplify a DNA fragment containing the gene encodingthe anti-hTfR antibody and isolate the gene encoding the high-affinityantibody.

It is possible to prepare cDNA or phage DNA from the above cells such asthe hybridoma cells producing the high-affinity antibody to anti-hTfR,or from the above phage particles retaining high-affinity antibody toanti-hTfR, and perform PCR or the like using it as a template to amplifyand isolate a DNA fragment containing the gene encoding the whole orpart of the anti-hTfR antibody light chain, the anti-hTfR antibody heavychain, or a single-chain antibody, as an anti-hTfR antibody. In the samemanner, it is also possible to perform PCR or the like to amplify andisolate a DNA fragment containing the gene encoding the whole or part ofthe light chain variable region of the anti-hTfR antibody, or a DNAfragment containing the gene encoding the whole or part of the heavychain variable region of the anti-hTfR antibody.

A high-affinity anti-hTfR antibody can be obtained by incorporating thewhole or part of the gene encoding the light chain and the heavy chainof this high-affinity anti-hTfR antibody into an expression vector,transforming host cells such as mammalian cells with this expressionvector, and culturing the obtained transformant cells. Using thenucleotide sequence of the isolated gene encoding the anti-hTfRantibody, it is also possible to translate the amino acid sequence ofthe anti-hTfR antibody, and artificially synthesize a DNA fragmentencoding the same amino acid sequence. In artificially synthesizing aDNA fragment, the expression level of the anti-hTfR antibody in the hostcells can be enhanced by proper selection of the codons.

In order to introduce a mutation such as substitution, deletion,addition and the like into the amino acid sequence of the originalanti-hTfR antibody, a mutation may be introduced as desired into thegene encoding the anti-hTfR antibody contained in the isolated DNAfragment. Though the gene encoding the mutated anti-hTfR antibody has ahomology preferably not lower than 80%, more preferably not lower than90%, to the original gene, there is no particular limitation as to thelevel of homology. By introducing a mutation into the amino acidsequence so as to modify the number or the type of sugar chains bound tothe anti-hTfR antibody, it is also possible to enhance the stability ofthe anti-hTfR antibody in the body.

When introducing a mutation into the gene encoding the whole or part ofthe light chain variable region of the anti-hTfR antibody, the gene thusmutated has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, to the original gene, though there is noparticular limitation as to the level of homology. When replacing one ormore amino acids of the amino acid sequence of the light chain variableregion with other amino acids, the number of amino acids to be replacedis preferably 1 to 10, more preferably 1 to 5, still more preferably 1to 3, and even more preferably 1 or 2. When deleting one or more aminoacids of the amino acid sequence of the light chain variable region, thenumber of amino acid to be deleted is preferably 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. A combined mutation of these substitution and deletion of aminoacids can also be carried out. When adding one or more amino acids tothe light chain variable region, they may be added inside, or on theN-terminal side or C-terminal side of, the amino acid sequence of thelight chain variable region, and the number of amino acids added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, and even more preferably 1 or 2. A combined mutation of theseaddition, substitution, and deletion of amino acids can also be carriedout. The amino acid sequence of the light chain variable region thusmutated has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, still more preferably not lower than 95%to the amino acid sequence of the original light chain variable region.In particular, when replacing one or more amino acids of the amino acidsequence of CDR with other amino acids, the number of amino acidreplaced is preferably 1 to 5, more preferable 1 to 3, still morepreferably 1 or 2. When deleting one or more amino acid of the aminoacid sequence of CDR, the number of amino acids to be deleted ispreferably 1 to 5, more preferably 1 to 3, still more preferably 1 or 2.A combined mutation of these substitution and deletion of the amino acidcan also be carried out. When adding one or more amino acids, they maybe added inside, or on the N-terminal side or C-terminal side of, theamino acid sequence, and the number of amino acids added is preferably 1to 5, more preferably 1 to 3, still more preferably 1 or 2. A combinedmutation of these addition, substitution, and deletion of amino acidscan also be carried out. The amino acid sequence of respective mutatedCDR has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, and still more preferably not lower than95% to the amino acid sequence of the original CDR.

When introducing mutation into the gene encoding the whole or part ofthe heavy chain variable region of the anti-hTfR antibody, the gene thusmutated has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, to the original gene, though there is noparticular limitation as to the level of homology. When replacing one ormore amino acids of the amino acid sequence of the heavy chain variableregion with other amino acids, the number of amino acids to be replacedis preferably 1 to 10, more preferably 1 to 5, still more preferably 1to 3, and even more preferably 1 or 2. When deleting one or more aminoacids of the amino acid sequence of the heavy chain variable region, thenumber of amino acids to be deleted is preferably 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. A combined mutation of these substitution and deletion of aminoacids can also be carried out. When adding one or more amino acid to theheavy chain variable region, they may be added inside, or on theN-terminal side or C-terminal side of, the amino acid sequence of theheavy chain variable region, and the number of amino acids added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, and even more preferably 1 or 2. A combined mutation of theseaddition, substitution, and deletion of amino acids can also be carriedout. The amino acid sequence of the heavy chain variable region thusmutated has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, still more preferably not lower than 95%to the amino acid sequence of the original heavy chain variable region.In particular, when replacing one or more amino acids of the amino acidsequence of CDR with other amino acids, the number of amino acidreplaced is preferably 1 to 5, more preferable 1 to 3, still morepreferably 1 or 2. When deleting one or more amino acid of the aminoacid sequence of CDR, the number of amino acids to be deleted ispreferably 1 to 5, more preferably 1 to 3, still more preferably 1 or 2.A combined mutation of these substitution and deletion of the amino acidcan also be carried out. When adding one or more amino acids, they maybe added inside, or on the N-terminal side or C-terminal side of, theamino acid sequence, and the number of amino acids added is preferably 1to 5, more preferably 1 to 3, still more preferably 1 or 2. A combinedmutation of these addition, substitution, and deletion of amino acidscan also be carried out. The amino acid sequence of respective mutatedCDR has a homology that is preferably not lower than 80%, morepreferably not lower than 90%, and still more preferably not lower than95% to the amino acid sequence of the original CDR.

A mutation may be introduced into both the variable regions of the lightchain and the heavy chain of the anti-hTfR antibody, by combining theabove mutation into the light chain variable region of the anti-hTfRantibody and the above mutation into the heavy chain variable region ofthe anti-hTfR antibody.

Examples of the above mentioned substitution of one or more amino acidsin the amino acid sequence of the light chain and the heavy chain of theanti-hTfR antibody include amino acids classified into the same groups,such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids(Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids(Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having ahydroxy group (Ser, Thr). It is expected that the substitution betweensuch analogous amino acids does not bring about any change to thephenotype of the protein (i.e., it is conservative amino acidsubstitution). However, the amino acid sequence of the framework region3 (SEQ ID NO: 83) of hTfR of antibody No. 3 (not published at the pointof filing of the present application) previously found by the presentinventor and confirmed to have the same type of effect as in the presentapplication has Trp at position 17 thereof. By contrast, the heavy chainframework region 3 of anti-hTfR antibody No. 3N of the present inventionhas the amino acid sequence set forth as SEQ ID NO: 68 in which theposition 17 from the N-terminal side thereof is substituted by Leu.Further, in the amino acid sequence of CDR1, Thr at position 5 of SEQ IDNO: 12 is substituted by Met, as shown in SEQ ID NO: 66. Trp and Leu donot have the above mentioned analogous relationship, and Thr and Met donot have the above mentioned analogous relationship. Nonetheless, asmentioned later, antibody No. 3N containing the substitution has,unexpectedly, the same type of effect as that of antibody No. 3 andexhibits superior effects.

Besides, in the case where a mutation is introduced into the anti-hTfRantibody by adding one or more amino acids to the C-terminus or theN-terminus, if the anti-hTfR antibody and a different protein (A) arefused placing the added amino acids between them, the added amino acidsconstitutes part of a linker. A detailed explanation will be given lateron a linker that is placed between the anti-hTfR antibody and a protein(A) in the fusion protein of the anti-hTfR antibody and the protein (A).

The anti-hTfR antibody obtained by culturing the cells selected by theabove methods and the like, as producing an anti-hTfR antibody that hasa relatively the high-affinity anti-hTfR antibody to hTfR, and theanti-hTfR antibody obtained by expression of the gene encoding ahigh-affinity anti-hTfR antibody, may be modified by introducing amutation into their amino acid sequences, such as substitution,deletion, addition to give them desired properties. Introduction of amutation into the amino acid sequence of the anti-hTfR antibody may beperformed by introducing a mutation into the gene corresponding to theamino acid sequence.

The affinity of an anti-hTfR antibody to hTfR can be adjusted as desiredby introduction of a mutation, such as substitution, deletion, andaddition, into the amino acid sequence of a variable region of theantibody. For example, if an antibody has such a high affinity to itsantigen that leads to too low a dissociation constant in water, there isa possibility that the antibody could, after administered to the body,fail to dissociate from the antigen, thereby leading to a functionaldisadvantage. In such a case, a most preferable antibody suitable to agiven purpose can be obtained by introducing a mutation into thevariable region of the antibody so as to adjust its dissociationconstant stepwise to 2 to 5 times, 5 to 10 times, 10 to 100 times, andso on, that of the original antibody. Conversely, the dissociationconstant can be adjusted stepwise to ½ to ⅕ times, ⅕ to 1/10 times, 1/10to 1/100 times, and so on, that of the original antibody, by introducinga mutation.

Introduction of a mutation such as substitution, deletion and additionto the amino acid sequence of the anti-hTfR antibody can be performed byintroducing a mutation into certain positions of the nucleotide sequenceof the gene either, for example, by PCR or the like using the geneencoding the anti-hTfR antibody as a template, or by random introductionof a mutation.

Introduction of a mutation into the amino acid sequence of the anti-hTfRantibody for adjusting the affinity of the antibody to hTfR can becarried out by, for example, incorporating a gene encoding the anti-hTfRantibody as a single-chain antibody into a phagemid, preparing with thisphagemid a phage with expressed single-chain antibody on the surface ofits capsid, letting the phage multiply while introducing a mutation intothe gene encoding the single-chain antibody by application of a mutagenor the like, and selecting, from the multiplied phage, a phageexpressing a single-chain antibody having a desired dissociationconstant either by the method described above or by purification usingan antigen column under a certain condition.

The antibodies having a relatively high-affinity to hTfR obtained by theabove-mentioned method of selecting the cells producing a high affinityantibody, are those whose dissociation constant (K_(D)) with hTfR asmeasured by the method described in Example 7 is preferably not greaterthan 1×10⁻⁸ M, more preferably not greater than 1×10⁻⁹ M, still morepreferably not greater than 1×10⁻¹⁰ M, and even more preferably notgreater than 1×10⁻¹¹ M. For example, those having a dissociationconstant of 1×10⁻¹³ M to 1×10⁻⁹ M, or 1×10⁻¹³ M to 1×10⁻¹⁰ M arepreferable. The same also applies if the antibodies are single-chainantibodies. Once an antibody is obtained, it can be modified as desiredby, e.g., introducing a mutation to give it a desired property.

Antibody having affinity both to human and monkey TfRs can be obtainedby selection of antibodies having affinity to monkey TfR from theanti-hTfR antibody that have been obtained as described above. Selectionof antibodies having affinity to monkey TfR can be carried out by, forexample, ELISA using a recombinant monkey TfR which is preparedutilizing recombinant DNA technologies. In such an ELIZA, a recombinantmonkey TfR is added to a plate and held by it, and contacted by theanti-hTfR antibody, and, after removal of antibody unbound to therecombinant monkey TfR from the plate, the amount of the antibody heldby the plate is measured. The higher the affinity of it to therecombinant monkey hTfR is, the greater the amount of the antibody heldby the plate becomes, and therefore. Consequently, the antibodycorresponding to the plate which held the greater amount of antibody canbe selected as the antibody having affinity to monkey TfR. Here, theterm “monkey” is preferably classified as simians except human, morepreferably as Cercopithecidae, still more preferably as macaques, andfor example crab-eating monkey or Rhesus monkey, among which crab-eatingmonkey is convenient for use in examination.

An antibody having affinity both to human and monkey hTfRs offers anadvantage that it allows pharmacokinetic observation of the antibodyadministered to the body using a monkey. For example, if a medical drugis being developed utilizing such an anti-hTfR antibody of the presentinvention, the progress of its development can be remarkablyaccelerated, for its pharmacokinetic study can be performed using amonkey.

In the present invention, an antibody having a relatively high affinityto hTfR and also having affinity to monkey TfR exhibits a dissociationconstant with human and monkey TfRs as follows, in particular, asmeasured by the method described in Example 7:

(a) dissociation constant with hTfR: preferably not greater than 1×10⁻¹⁰M, more preferably not greater than 2.5×10⁻¹¹ M, still more preferablynot greater than 5×10⁻¹² M, and even more preferably not greater than1×10⁻¹² M, and

(b) dissociation constant with monkey TfR: preferably not greater than1×10⁻⁹ M, more preferably not greater than 5×10⁻¹⁰ M, and still morepreferably not greater than 1×10⁻¹⁰ M, for example, not greater than7.5×10⁻¹¹ M.

For example, the dissociation constants with hTfR and monkey TfR are notgreater than 1×10⁻¹⁰ M and not greater than 1×10⁻⁹ M, not greater than1×10⁻¹¹ M and not greater than 5×10⁻¹⁰ M, not greater than 5×10⁻¹² M andnot greater than 1×10⁻¹⁰ M, not greater than 5×10⁻¹² M and not greaterthan 7.5×10⁻¹¹ M, not greater than 1×10⁻¹² M and not greater than1×10⁻¹⁰ M, or not greater than 1×10⁻¹² M and not greater than 7.5×10⁻¹¹M, respectively. In this context, although there is no particularlydefinite lower limit on the dissociation constant with human TfR, it canbe, for example, 5×10⁻¹³ M or 1×10⁻¹³ M. Although there is noparticularly definite lower limit on the dissociation constant withmonkey TfR, it can be, for example, 1×10⁻¹¹ M or 1×10⁻¹² M. The samealso applies if the antibody is a single-chain antibody.

If an antibody having a relatively high-affinity to hTfR and obtained bythe above method in which those cells producing a high affinity antibodywere selected, is an antibody of a non-human animal, it may be convertedto a humanized antibody. A humanized antibody is an antibody produced byusing an amino acid sequence of part of the variable region (e.g., thewhole or part of the CDRs) of a non-human animal antibody, and replacinga proper region of a human antibody with the sequence (which isimplanted in the human antibody) while maintaining the specificity tothe antigen. Examples of humanized antibodies include an antibodyproduced by replacing the three complementarity determining regions(CDRs) in the immunoglobulin light chain and the three complementaritydetermining regions (CDRs) in the immunoglobulin heavy chain, bothconstituting a human antibody, with CDRs of a non-human mammal. Thoughthere is no particular limitation as to the biological species fromwhich the CDRs to be incorporated into the human antibody are derived solong as it is a non-human mammal, it preferably is a mouse, rat, rabbit,horse, and non-human primate, more preferably a mouse and rat, and stillmore preferably a mouse. However, it may be an antibody in which part ofa human antibody is replaced with part of a different human antibody.

Methods for preparation of humanized antibody are well known in the artand the most common is a method in which the amino acid sequence of thecomplementarity determining regions (CDRs) in the variable region of ahuman antibody is replaced with the CDRs of an antibody of non-humanmammal, as devised by Winter et al. (Verhoeyen M. Science. 239.1534-1536 (1988)). It is also well known that in some cases,corresponding part of an acceptor human antibody needs to be replacednot only with the CDRs of the non-human mammalian antibody but alsoamino acid sequences occurring in regions outside the CDRs that play arole either in maintaining the structure of the CDRs or in binding tothe antigen, in order to reproduce the activity that the donor antibodyoriginally possesses (Queen C. Proc. Natl. Acad. Sci. USA. 86.10029-10033 (1989)). Here, the regions outside the CDRs are calledframework (FR) regions.

Both the heavy chain and light chain variable regions of an antibodycomprise four framework regions 1 to 4 (FR1 to FR4). FR1 is a regionadjacent to CDR1 on the N-terminal side thereof, and consists of anamino acid sequence from the N-terminus in each peptide constituting theheavy chain and the light chain to an amino acid adjacent to theN-terminus of CDR1 thereof FR2 consists of an amino acid sequencebetween CDR1 and CDR2 in each peptide constituting the heavy chain andthe light chain. FR3 consists of an amino acid sequence between CDR2 andCDR3 in each peptide constituting the heavy chain and the light chain.FR4 consists of an amino acid sequence from an amino acid adjacent tothe C-terminus of CDR3 to the C-terminus of the variable region.However, in the present invention, which is not limited thereby, aregion excluding 1 to 5 N-terminal side amino acids and/or 1 to 5C-terminal side amino acids in each FR region mentioned above may beused as the framework region.

Preparation of humanized antibody involves processes of implanting theCDRs (and their neighboring FRs, as the case may be) of non-humanmammalian antibody in place of the CDRs (and their neighboring FRs, asthe case may be) in the variable region of a human antibody. In suchprocesses, the starting framework region of the variable region of ahuman antibody can be obtained from a public DNA database and the likewhich includes germ line antibody genes. For example, germ line DNAsequences, as well as amino acid sequences, of human heavy chain andlight chain variable regions can be selected from “VBase” human germlinedatabase (available in the Internet, at www.mrc-cpe.cam.ac.uk/vbase).Besides, they can be selected from publicized DNA sequences and aminoacid sequences described in literatures, such as “Kabat E A. Sequencesof Proteins of Immunological Interest, 5th Ed., U.S. Depaitment ofHealth and Human Services, NIH Publication No. 91-3242 (1991”);“Tomlinson I M. J. fol. Biol. 227. 776-98 (1992)”; and “Cox J P L. Eur.J Immunol 24:827-836 (1994)”.

As aforementioned, in a humanized antibody, the regions of a non-humanmammalian animal antibody to be implanted into the variable regions ofthe original human antibody generally include CDRs themselves, or CDRsand their neighboring part of FRs. However, such FRs implanted togetherwith CDRs also play a role either in maintaining the structure of theCDRs or in binding to the antigen, thus having a substantial function indetermining the complementarity of an antibody, and the term “CDR” inthe present invention, therefore, refers to such regions that are, orcould be, taken from a non-human mammalian animal antibody and implantedinto a humanized antibody, in preparing a humanized antibody. Thus, aregion generally considered to be in a FR region is included in a CDR inthe present invention as far as it takes part either in maintaining thestructure of the CDR or in binding to the antigen, and is thusconsidered to have a substantial function in determining thecomplementarity of the antigen.

The anti-hTfR antibody of the present invention, when administered tothe body, e.g., by intravenous injection, efficiently binds to hTfRoccurring on the endothelial cells of the capillaries in the brain. Theantibody bound to the hTfR is taken into the brain across theblood-brain barrier by such mechanisms as endocytosis, and transcytosis.Therefore, by binding them to the anti-hTfR antibody of the presentinvention, those proteins, low-molecular-weight compounds and the likethat need to be brought into function in the brain, can be efficientlydelivered into the brain across the blood-brain barrier. Further, theanti-hTfR antibody of the present invention can, after passing throughthe blood-brain barrier, can reach the cerebral parenchyma, andnerve-like cells in the hippocampus; Purkinje cells and the like of thecerebellum or at least one of them. And it is also expected that itreaches to the nerve-like cells in the striatum of the cerebrum; and thenerve-like cells in the substantia nigra of the mesencephalon.Therefore, it is possible to make one of those proteins,low-molecular-weight compounds and the like, which could act on suchtissues or cells, reach the tissues or cells, by binding it to theanti-hTfR antibody of the present invention.

The anti-hTfR antibody of the present invention can be an effectivemeans to make those compounds (proteins, low-molecular-weight compoundsand the like) transfer from the blood into the brain and function there,which compounds otherwise cannot pass through the blood-brain barrierwhen intravenous administered and therefore cannot or can hardly exhibittheir physiological or pharmacological functions in the brain. Inparticular, the anti-hTfR antibody of the present invention can, afterpassing through the blood-brain barrier, reach the cerebral parenchyma,and nerve-like cells in the hippocampus; Purkinje cells and the like ofthe cerebellum or at least one of them. And it is also expected that itreaches to the nerve-like cells in the striatum of the cerebrum; as wellas to the nerve-like cells in the substantia nigra of the mesencephalon.Therefore, it is possible to make those compounds function or augmenttheir function, in those tissues or cells in the brain by administeringthose compounds in a combined form with the anti-hTfR antibody molecule,parenterally, e.g., intravenously.

For binding an anti-hTfR antibody to such compounds (proteins,low-molecular-weight compounds and the like), a method is available tobind them together via a non-peptide linker or a peptide linker. Asnon-peptide linkers, there can be used polyethylene glycol,polypropylene glycol, copolymer of ethylene glycol and propylene glycol,polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ether, biodegradable polymer, polymerized lipid, chitins, andhyaluronic acid, or derivatives thereof, or combinations thereof. Apeptide linker is a peptide chain consisting of 1 to 50 amino acidslinked by peptide bonds or a derivative thereof, whose N-terminus andC-terminus are to be covalently bonded either to an anti-hTfR antibodyor a compound such as protein, low-molecular-weight compound and thelike, respectively, to bind the anti-hTfR antibody to such a compoundlike protein, low-molecular-weight compound.

In particular, a conjugate which is formed by binding the anti-hTfRantibody of the present invention to a desired different protein (A) viaPEG as a non-peptide linker, is designated “anti-hTfRantibody-PEG-protein”. An anti-hTfR antibody-PEG-protein can be preparedby first binding the anti-hTfR antibody to PEG to form anti-hTfRantibody-PEG, and then binding the anti-hTfR antibody-PEG to thedifferent protein (A). Alternatively, an anti-hTfR antibody-PEG-proteincan be prepared by first binding the different protein (A) to PEG toform “protein-PEG”, and then binding the “protein-PEG” to the anti-hTfRantibody. In order to bind PEG to the anti-hTfR antibody and thedifferent protein (A), a PEG is employed which is modified with suchfunctional groups as carbonate, carbonylimidazole, active ester ofcarboxylic acid, azlactone, cyclic imide thione, isocyanate,isothiocyanate, imidate, aldehyde or the like. Such a functional groupintroduced to PEG reacts mainly with amino groups in the anti-hTfRantibody and a different protein (A) to covalently bind PEG to the hTfRantibody and a different protein (A). Though there is no particularlimitation as to the molecular weight and the configuration of PEGemployed here, its mean molecular weight (MW) is as follows: preferablyMW=500 to 60000, more preferably MW=500 to 20000. For example, such PEGwhose mean molecular weight is about 300, about 500, about 1000, about2000, about 4000, about 10000, about 20000, and the like. PEG ispreferably used as a non-peptide linker. The anti-hTfR antibody can bebound to a desired low-molecular-weight compound in the same manner asabove.

For example, “anti-hTfR antibody-PEG” can be prepared by mixing theanti-hTfR antibody with a polyethylene glycol having aldehyde groups asfunctional groups (ALD-PEG-ALD) so that the molar ratio of ALD-PEG-ALDto the antibody is 11, 12.5, 15, 110, 120 and the like, and then addingto the mixture a reducing agent such as NaCNBH₃ to let a reaction takeplace. Then, by reacting “anti-hTfR antibody-PEG” with a differentprotein (A) in the presence of a reducing agent such as NaCNBH₃,“anti-hTfR antibody-PEG-protein” is obtained. On the contrary, it isalso possible to obtain “anti-hTfR antibody-PEG-protein” by firstbinding a different protein (A) to ALD-PEG-ALD to prepare “protein-PEG”,and then binding the “protein-PEG” to the anti-hTfR antibody.

The anti-hTfR antibody and a different protein (A) can also be boundtogether through peptide bonds by linking the anti-hTfR antibody heavychain or light chain, on the C-terminal side or the N-terminal sidethereof, either via a linker sequence or directly, to the N-terminus orthe C-terminus of the different protein (A), respectively. Thus thefusion protein between the anti-hTfR antibody and a different protein(A) can be obtained by incorporating into a mammalian expression vectora DNA fragment in which a cDNA encoding the different protein (A) isplaced in-frame directly, or via a DNA fragment encoding a linkersequence, on the 3′-end or 5′-end side of a cDNA encoding the heavychain or light chain of the anti-hTfR antibody, and culturing mammaliancells into which the above expression vector has been introduced. Wherethe DNA fragment encoding a different protein (A) is linked to the heavychain, a mammalian expression vector in which a cDNA fragment encodingthe anti-hTfR antibody light chain is also introduced into the same hostcells, whereas if DNA fragment encoding a different protein (A) islinked to the light chain, a mammalian expression vector in which a cDNAfragment encoding the anti-hTfR antibody heavy chain is alsoincorporated into the same host cells. In the case where the anti-hTfRantibody is a single-chain antibody, the fusion protein comprising theanti-hTfR antibody and a different protein (A) combined can be obtainedby incorporating, into an expression vector (for eukaryotic cells suchas mammalian and yeast, or for prokaryotic cells such as E. coli.), aDNA fragment which is formed by linking the cDNA encoding a differentprotein (A), on the 5′-end side or on the 3′-end side thereof, directlyor via a DNA fragment encoding a linker sequence, to the cDNA encodingthe single-chain anti-hTfR antibody, and allowing the fusion protein beexpressed in those cells into which the expression vector has beenintroduced.

In a fusion protein of the type in which a different protein (A) islinked to the anti-hTfR antibody light chain on the C-terminal sidethereof, the anti-human transferrin receptor antibody comprises an aminoacid sequence including the whole or part of the light chain variableregion and an amino acid sequence including the whole or part of theheavy chain variable region, and the different protein (A) is linked tothe light chain of this anti-human transferrin receptor antibody on theC-terminal side thereof Here, the anti-hTfR antibody light chain and adifferent protein (A) may be linked together, directly or via a linker.

In a fusion protein of the type in which a different protein (A) islinked to the anti-hTfR antibody heavy chain on the C-terminal sidethereof, the anti-human transferrin receptor antibody comprises an aminoacid sequence including the whole or part of the light chain variableregion and an amino acid sequence including the whole or part of theheavy chain variable region, and the different protein (A) is linked tothe heavy chain of this anti-human transferrin receptor antibody on theC-terminal side thereof Here, the anti-hTfR antibody heavy chain and adifferent protein (A) may be linked together, directly or via a linker.

In a fusion protein of the type in which a different protein (A) islinked to the anti-hTfR antibody light chain on the N-terminal sidethereof, the anti-human transferrin receptor antibody comprises an aminoacid sequence including the whole or part of the light chain variableregion and an amino acid sequence including the whole or part of theheavy chain variable region, and the different protein (A) is linked tothe light chain of this anti-human transferrin receptor antibody on theN-terminal side thereof Here, the anti-hTfR antibody light chain and adifferent protein (A) may be linked together, directly or via a linker.

In a fusion protein of the type in which a different protein (A) islinked to the anti-hTfR antibody heavy chain on the N-terminal sidethereof, the anti-human transferrin receptor antibody comprises an aminoacid sequence including the whole or part of the light chain variableregion and an amino acid sequence including the whole or part of theheavy chain variable region, and the different protein (A) is linked tothe heavy chain of this anti-human transferrin receptor antibody on theN-terminal side thereof Here, the anti-hTfR antibody heavy chain and adifferent protein (A) may be linked together, directly or via a linker.

In the above, the linker sequence placed between the anti-hTfR antibodyand a different protein (A) may be a peptide chain consisting preferablyof 1 to 50, more preferably of 1 to 17, still more preferably of 1 to10, even more preferably of 1 to 5 amino acids, and in accordance withthe different protein (A) to be linked to the anti-hTfR antibody, thenumber of amino acids of the linker sequence may be adjusted to 1, 2, 3,1-17, 1-10, 10-40, 20-34, 23-31, 25-29, 27, etc., as desired. Thoughthere is no particular limitation as to amino acid sequence of thelinker sequence insofar as the anti-hTfR antibody linked by it retainsthe affinity to hTfR and a different protein (A) linked by the linkersequence also exhibit the protein's own physiological activity under aphysiological condition, the linker may preferably be composed ofglycine and serine. Examples or such linkers include one consisting of asingle amino acid either glycine or serine, the amino acid sequenceGly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequenceGly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequenceGly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequenceSer-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence which includes 1-10 or2-5 of any of those amino acid sequences consecutively linked. They havesequences consisting of 1-50, 2-17, 2-10, 10-40, 20-34, 23-31, 25-29, or27 amino acids. For example, those comprising the amino acid sequenceGly-Ser may preferably be used as linker sequences. Further, a linkersequence comprising 27 amino acids is preferably used that is composedof the amino acid sequence Gly-Ser followed by consecutively linked fivecopies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3).Still further, a linker sequence comprising 25 amino acids is alsopreferably used that is composed of consecutively linked five copies ofthe amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

In a fusion protein of the anti-hTfR antibody and a different protein(A), where the anti-hTfR antibody is a single-chain antibody, the aminoacid sequence including the whole or part of the immunoglobulin lightchain variable region and the amino acid sequence including the whole orpart of the immunoglobulin heavy chain variable region are linked,generally via a linker sequence. Insofar as the affinity of theanti-hTfR antibody to hTfR is retained, the amino acid sequence derivedfrom the light chain may be linked, on the C-terminal side thereof, to alinker sequence which in turn being linked, on the C-terminal sidethereof, to the amino acid sequence derived from the heavy chain or,conversely, the amino acid sequence derived from the heavy chain may belinked, on the C-terminal side thereof, to a linker sequence which inturn being linked, on the C-terminal side thereof, to the amino acidsequence derived from the light chain.

The linker sequence placed between the light chain and the heavy chainof the immunoglobulin is a peptide chain consisting preferably of 2-50,more preferably 8-50, still more preferably 10-30, even more preferably12-18 or 15-25, and for example 15 or 25 amino acids. Though there is nospecific limitation as to the linker sequence insofar as the anti-hTfRantibody made of the both chains which are linked via the linker retainsthe affinity to hTfR and a different protein (A) linked to the antibodyalso exhibits the protein's own physiological activity under aphysiological condition, the linker is preferably composed of glycine,or glycine and serine. Examples of such linkers include the amino acidsequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acidsequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQID NO:3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4),the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequencewhich includes 2-10 or 2-5 of any of these amino acid sequencesconsecutively linked. A preferred embodiment of such a linker sequencecomprises 15 amino acids consisting of consecutively linked three copiesof the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3).

In the case where the anti-hTfR antibody is a single-chain antibody, anexample of specific embodiments of the fusion protein between thehumanized anti-hTfR antibody of the present invention and a differentprotein (A) is a fusion protein consisting of the different protein (A)which is linked, on the C-terminal side thereof and via a first linkersequence consisting of 27 amino acids composed of the amino acidsequence Gly-Ser followed by consecutively linked five copies of theamino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to thesingle-chain antibody. An example of a preferred embodiment ofsingle-chain antibodies employed here is an antibody having the aminoacid sequence set forth as SEQ ID NO:60, which is composed of the aminoacid sequence of the anti-hTfR antibody No. 3N heavy chain variableregion set forth as SEQ ID NO:65 that is linked, at the C-terminusthereof and via a first linker sequence consisting of 15 amino acidsconsisting of consecutively linked three copies of the amino acidsequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to the anti-hTfR antibodylight chain variable region having the amino acid sequence set forth asSEQ ID NO:18, an antibody having the amino acid sequence set forth asSEQ ID NO:98, which is composed of the amino acid sequence of the Fabheavy chain of the anti-hTfR antibody No. 3N set forth as SEQ ID NO:61that is linked, at the C-terminus thereof and via a linker sequenceconsisting of 32 amino acids consisting of consecutively linked sixcopies of the amino acid sequence set forth asSEQ ID NO:3 followed bythe amino acid sequence of Gly-Gly, to the anti-hTfR antibody lightchain variable region having the amino acid sequence set forth as SEQ IDNO:23.

Where the anti-hTfR antibody is a single-chain antibody, such a fusionprotein can be produced by, for example, transforming host cells such asmammalian cells with an expression vector having an incorporated DNAfragment containing a nucleotide sequence encoding the fusion protein,and then culturing the host cells.

Besides, in the present invention, when a peptide chain includes aplurality of linker sequences, each of those linker sequences isdesignated, from the N-terminal side, the first linker sequence, thesecond linker sequence, and so on, for convenience.

In the case where the anti-hTfR antibody is Fab, an example of specificembodiments of the fusion protein between a humanized anti-hTfR antibodyand a different protein (A) of the present invention is a fusion proteinwhich is composed of the different protein (A) that is fused, on theC-terminal side thereof and via a linker sequence consisting of 27 aminoacids composed of Gly-Ser followed by consecutively linked five copiesof the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to aregion having the anti-hTfR antibody heavy chain variable region and theC_(H)1 region. Though part of the hinge region may be included inaddition to the C_(H)1 region here, the hinge region includes nocysteine residue which would form a disulfide bond between heavy chains.

In the case where the anti-hTfR antibody is Fab, an example of apreferred heavy chain is one having the amino acid sequence set forth asSEQ ID NO: 61. The amino acid sequence set forth as SEQ ID NO: 61 is Fabof the heavy chain of humanized anti-hTfR antibody No. 3N having theamino acid sequence set forth as SEQ ID NO: 66, and corresponds to aportion at positions 1 to 226 from the N-terminal side of the amino acidsequence set forth as SEQ ID NO: 66. Besides, a portion at positions 1to 118 from the N-terminus of SEQ ID NO: 61 corresponds to the variableregion (SEQ ID NO: 65), a portion at positions 119 to 216 corresponds tothe C_(H)1 region, and a portion at positions 217 to 226 corresponds tothe hinge region.

In the case where the anti-hTfR antibody is Fab, the Fc region ofdifferent IgG may be further introduced in the fusion protein. Byintroducing the Fc region in the fusion protein, it is possible toenhance the stability of the fusion protein in the body, such as inblood. Such a fusion protein with the Fc region introduced therein is,for example, the one in which a human IgG Fc region is linked, directlyor via a linker sequence, to the different protein (A) on the C-terminalside thereof, and the Fab heavy chain of the anti-human transferrinreceptor antibody is linked, directly or via a linker sequence, to thehuman IgG Fc region on the C-terminal side thereof.

The linker sequence between the different protein (A) and the human IgGFc region consists preferably of 1 to 50 amino acids. In this context,the number of amino acids is adjusted to 1-17, 1-10, 10-40, 20-34,23-31, 25-29, 27, etc., as desired. Though there is no particularlimitation as to amino acid sequence of the linker sequence, the linkermay preferably be composed of glycine and serine. Examples of suchlinkers include one consisting of a single amino acid either glycine orserine, the amino acid sequence Gly-Ser, the amino acid sequenceGly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3),the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), theamino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequencewhich has 1-10 or 2-5 of any of those amino acid sequences consecutivelylinked, and includes a sequence consisting of not more than 50 aminoacids or a sequence consisting of 2-17, 2-10, 10-40, 20-34, 23-31,25-29, or 25 amino acids. For example, a linker sequence comprising 25amino acids is preferably used that is composed of consecutively linkedfive copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3). The same also applies for the linker sequence between the human IgGFc region and the Fab heavy chain.

Besides, when introducing the human IgG Fc region, the human IgG Fcregion may be linked to either of the heavy chain or the light chain ofthe anti-human transferrin receptor antibody. Further, the antibody maybe other antigen-binding fragments including F(ab′)₂, F(ab′), and asingle-chain antibody.

There is no particular limitation as to the IgG type of the human IgG Fcregion to be introduced, and it may be any of IgG1 to IgG5. Further, thehuman IgG Fc region to be introduced may be the whole Fc region or maybe part thereof. A preferred embodiment of such a human IgG Fc region isone having the amino acid sequence set forth as SEQ ID NO: 70, which isthe whole region of human IgG1 Fc. Further, the amino acid sequence ofthe Fab heavy chain with the human IgG Fc region added thereto is onehaving the amino acid sequence set forth as SEQ ID NO: 71 in which thehuman IgG Fc region having the amino acid sequence set forth as SEQ IDNO: 70 is linked, via a linker sequence comprising 25 amino acids thatis composed of consecutively linked five copies of the amino acidsequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), to the N-terminal side ofthe amino acid sequence of the Fab heavy chain (SEQ ID NO: 61) of thehumanized anti-hTfR antibody 3N.

The fusion protein between an anti-hTfR antibody and a different protein(A) can be modified so as to have an affinity to albumin. The affinityto albumin can be attained by binding to the fusion protein a compound,a peptide, a protein or the like having an affinity to albumin. Thefusion protein, into which an affinity to albumin has been introduced,is at least partially bound to albumin, and circulated in blood. Albuminhas a function of stabilizing a protein bound thereto. Therefore, byintroducing an affinity to albumin, the in-blood half-life of the fusionprotein administered in a living body can be extended, so that thepharmacological effect of the fusion protein can be enhanced.Introduction of an affinity to albumin is more effective when theanti-hTfR antibody is an antibody lacking a Fc region contributing tostability of the antibody, e.g. Fab.

Introduction of an affinity to albumin is also effective when the fusionprotein between an anti-hTfR antibody and a different protein (A) showsimmunogenicity when administered in a living body. With the fusionprotein bound to albumin, a site of the fusion protein which showsimmunogenicity is inhibited from being presented to immune cells, sothat immunogenicity is reduced.

When an affinity to albumin is introduced into the fusion proteinbetween an anti-hTfR antibody and a different protein (A), the regioninto which the affinity is introduced may be any one of a light chain ofthe anti-hTfR antibody, a heavy chain of the anti-hTfR antibody, adifferent protein (A) and a linker region, or the affinity may beintroduced into two or more of these regions.

As peptides or proteins having an affinity to albumin, for example,there can be used, among others, peptides in which an albumin-bindingdomain of a protein derived from Streptococcus strain G418 (Alm T.Biotechnol J. 5. 605-17 (2010)) having an amino acid sequence set forthas SEQ ID NO: 74 are modified so as to have alkali resistance. Forbinding together a peptide or protein having an affinity to albumin(albumin-affinitive peptide) and a fusion protein between an anti-hTfRantibody and a different protein (A) (anti-hTfR antibody-protein (A)fusion protein), a method is available to bind them together via anon-peptide linker or a peptide linker. As non-peptide linkers, therecan be used polyethylene glycol, polypropylene glycol, copolymer ofethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinylalcohol, polysaccharides, dextran, polyvinyl ether, biodegradablepolymer, lipid polymer, chitins, and hyaluronic acid, or derivativesthereof, or combinations thereof. The peptide linker is a peptide chainconsisting of 1 to 50 amino acids linked by peptide bonds or aderivative thereof, whose N-terminus and C-terminus are covalentlybonded either to an albumin-affinitive peptide or a fusion protein,respectively, to bind the albumin-affinitive peptide and the fusionprotein together.

In particular, a conjugate, which is formed by binding thealbumin-affinitive peptide to the fusion protein between an anti-hTfRantibody and a different protein (A) via PEG as a non-peptide linker, isdesignated “albumin-affinitive peptide-PEG-protein (A) fusion protein”.An albumin-affinitive peptide-anti-hTfR antibody-protein (A) fusionprotein can be prepared by binding the albumin-affinitive peptide to PEGto form an albumin-affinitive peptide-PEG, and then binding thealbumin-affinitive peptide-PEG to an anti-hTfR antibody-protein (A)fusion protein. Alternatively, an albumin-affinitive peptide-anti-hTfRantibody-protein (A) fusion protein can be prepared by binding ananti-hTfR antibody-protein (A) fusion protein to PEG to form ananti-hTfR antibody-protein (A) fusion protein, and then binding theanti-hTfR antibody-protein (A) fusion protein-PEG to analbumin-affinitive peptide. In order to bind PEG to thealbumin-affinitive peptide and the anti-hTfR antibody-protein (A) fusionprotein, a PEG is employed which is modified with such functional groupsas carbonate, carbonylimidazole, active ester of carboxylic acid,azlactone, cyclic imide thione, isocyanate, isothiocyanate, imidate,aldehyde or the like. Such a functional group introduced to PEG reactsmainly with amino groups in the molecules of the albumin-affinitivepeptide and the anti-hTfR antibody-protein (A) fusion protein tocovalently bind PEG to the albumin-affinitive peptide and the anti-hTfRantibody-protein (A) fusion protein. Though there is no particularlimitation as to the molecular weight and the configuration of PEGemployed here, its mean molecular weight (MW) is as follows: preferablyMW=500 to 60000, more preferably MW=500 to 20000. For example, such PEGwhose mean molecular weight is about 300, about 500, about 1000, about2000, about 4000, about 10000, about 20000, and the like. PEG ispreferably used as a non-peptide linker.

For example, an albumin-affinitive peptide-PEG can be prepared by mixingan albumin-affinitive peptide with an aldehyde group-modified PEG(ALD-PEG-ALD) in such a manner that the molar ratio of the modified PEGto the albumin-affinitive peptide is 11, 12.5, 15, 110, 120 or the like,and then adding to the mixture a reducing agent such as NaCNBH₃ to let areaction take place. Then, by reacting the albumin-affinitivepeptide-PEG with an anti-hTfR antibody-protein (A) fusion protein in thepresence of a reducing agent such as NaCNBH₃, an albumin-affinitivepeptide-PEG-anti-hTfR antibody-protein (A) fusion protein is obtained.On the contrary, it is also possible to obtain an albumin-affinitivepeptide-PEG-anti-hTfR antibody-protein (A) fusion protein by firstbinding an anti-hTfR antibody-protein (A) fusion protein to ALD-PEG-ALDto prepare an anti-hTfR antibody-protein (A) fusion protein-PEG, andthen binding the fusion protein-PEG to an albumin-affinitive peptide.

It is also possible to fuse the anti-hTfR antibody-protein (A) fusionprotein to the albumin-affinitive peptide. The fusion protein (anti-hTfRantibody-protein (A) fusion protein-albumin-affinitive peptide) can beobtained by incorporating into a mammalian expression vector a DNAsegment in which a cDNA encoding the albumin-affinitive peptide isplaced in-frame directly, or via a DNA segment encoding a linkersequence, on the 3′-end or 5′-end side of a cDNA encoding the heavychain (including a fusion protein between the heavy chain and theprotein (A)) or the light chain (including a fusion protein between thelight chain and the protein (A)) of the anti-hTfR antibody-protein (A)fusion protein, and culturing mammalian cells into which the expressionvector has been introduced. When a DNA fragment encoding analbumin-affinitive peptide is bound to the heavy chain (or fusionprotein between the heavy chain and the protein (A)), a mammalianexpression vector in which a cDNA segment encoding a fusion proteinbetween the light chain constituting the anti-hTfR antibody and theprotein (A) (or light chain) has been incorporated is also introducedinto the same host cells, whereas when a DNA segment encoding analbumin-affinitive peptide is bound to the light chain (or fusionprotein between the light chain and the protein (A)), a mammalianexpression vector in which a cDNA segment encoding a fusion proteinbetween the heavy chain of the anti-hTfR antibody and the protein (A)(or heavy chain) has been incorporated is also introduced into the samehost cells. That is, the albumin-affinitive peptide may be bound eitheron the N-terminal or C-terminal side of the heavy chain (including afusion protein between the heavy chain and the protein (A)) or the lightchain (including a fusion protein between the light chain and theprotein (A)) of the anti-hTfR antibody-protein (A) fusion protein, butwhen the protein (A) is bound to the heavy chain of the anti-hTfRantibody on the N-terminal side, it is preferable to bind thealbumin-affinitive peptide on the C-terminal side of the anti-hTfRantibody, and it is particularly preferable to bind thealbumin-affinitive peptide on the C-terminal side of the heavy chain.

The anti-hTfR antibody-protein (A) fusion protein can be fused with thealbumin-affinitive peptide directly, or via a linker sequence. Here, thelinker sequence consists of preferably 1 to 50 amino acids. Here, thenumber of amino acids is adjusted to 1-17, 1-10, 10-40, 20-34, 23-31,25-29, 27, etc., as appropriate. Though there is no limitation as to theamino acid sequence of the linker sequence, the linker sequencepreferably consists of glycine and serine. Examples of the linkersinclude those having sequences consisting of 1-50, 2-17, 2-10, 10-40,20-34, 23-31, 25-29 or 25 amino acids, where the linker consists of oneof amino acids: glycine and serine, the amino acid sequence: Gly-Ser,the amino acid sequence Gly-Gly-Ser, the amino acid sequenceGly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequenceGly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), the amino acid sequenceSer-Gly-Gly-Gly-Gly (SEQ ID NO: 5), or a sequence which includes 1 to 10or 2 to 5 of any of those amino acid sequences consecutively linked. Forexample, a linker sequence comprising total 15 amino acids may bepreferably used which consists of three consecutively linked copies ofamino acid sequence: Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

The binding affinity to albumin of the anti-hTfR antibody-protein (A)fusion protein in which an albumin-affinitive peptide has beenintroduced is preferably not greater than 1×10⁻⁷ M, more preferably notgreater than 5×10⁻⁷ M, still more preferably not greater than 1×10⁻⁸ M,even more preferably not greater than 1×10⁻⁹ M as measured by bio-layerinterferometry as described in Example 7.

The Fab antibody can be stabilized in blood even by a method other thanintroduction of a Fc region or an albumin-affinitive peptide. Forexample, the Fab antibody can be stabilized by PEG-modifying the Fabantibody itself or a fusion between the Fab antibody and a differentprotein. Such a method is generally carried out in the field of proteinpharmaceuticals, and PEGylated erythropoietin, interferon, etc. havebeen put into practical use as pharmaceutical products. The Fab antibodycan also be stabilized by introducing a mutation into the Fab antibody.For example, the Fab antibody can be stabilized by replacing methionineat position 4 from the N-terminal side of the light chain with leucine.However, the method for introducing a mutation is not limited thereto,and a mutation may be introduced into the heavy chain. In addition, themethod for stabilizing the Fab antibody is not limited to thosedescribed above, and all well known methods can be utilized.

Though there is no particular limitation as to the different protein (A)to be linked to the anti-hTfR antibody, it is a protein that can exhibitits physiological activity in the body, and in particular, such aprotein that needs to get inside the brain and exhibit its functionthere but, due to its inability to pass through the blood-brain barrieras it is, cannot be expected to function in the brain if simplyadministered intravenously. Examples of such proteins include lysosomalenzymes such as nerve growth factor (NGF), α-L-iduronidase (IDUA),iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase,GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B,N-acetylglucosamine-1-phosphotransferase, α-mannosidase (LAMAN),β-mannosidase, galactosylceramidase (GALC), saposin C, arylsulfatase A(ARSA), α-L-fucosidase (FUCA1), aspartylglucosaminidase,α-N-acetylgalactosaminidase, acidic sphingomyelinase (ASM),α-galactosidase A, β-glucuronidase (GUSB), heparan N-sulfatase (SGSH),α-N-acetylglucosaminidase (NAGLU), acetyl CoA:α-glucosaminideN-acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, acidceramidase (AC), amylo-1,6-glucosidase, sialidase,aspartylglucosaminidase, palmitoyl protein thioesterase 1 (PPT-1),tripeptidyl-peptidase 1 (TPP-1), hyaluronidase 1, acidic α-glucosidase(GAA), CLN1, and CLN2, and the like.

The nerve growth factor (NGF) linked to the anti-hTfR antibody can beused as a therapeutic agent for dementia in Alzheimer's disease;α-L-iduronidase (IDUA) linked to the anti-hTfR antibody as a therapeuticagent for central nervous system disorders in Hurler syndrome orHurler-Scheie syndrome; iduronate 2-sulfatase (IDS) linked to theanti-hTfR antibody as a therapeutic agent for central nervous systemdisorders in Hunter syndrome; glucocerebrosidase (GBA) as a therapeuticagent for central nervous system disorders in Gaucher's disease;β-galactosidase as a therapeutic agent for central nervous systemdisorders in GM1 gangliosidosis Types 1-3; GM2 activator protein as atherapeutic agent for central nervous system disorders inGM2-gangliosidosis, AB variant; β-hexosaminidase A as a therapeuticagent for central nervous system disorders in Sandhoff's disease andTay-Sachs disease; β-hexosaminidase B as a therapeutic agent for centralnervous system disorders in Sandhoff's disease;N-acetylglucosamine-1-phosphotransferase as a therapeutic agent forcentral nervous system disorders in I-cell disease; α-mannosidase(LAMAN) as a therapeutic agent for central nervous system disorders inα-mannosidosis; β-mannosidase as a therapeutic agent for central nervoussystem disorders in β-mannosidosis; galactosylceramidase (GALC) as atherapeutic agent for central nervous system disorders in Krabbedisease; saposin C as a therapeutic agent for central nervous systemdisorders in Gaucher's disease-like storage disease; arylsulfatase A(ARSA) as a therapeutic agent for central nervous system disorders inmetachromatic white matter degeneration (metachromatic leukodystrophy);α-L-fucosidase (FUCA1) as a therapeutic agent for central nervous systemdisorders in fucosidosis; aspartylglucosaminidase as a therapeutic agentfor central nervous system disorders in aspartylglucosaminuria;α-N-acetylgalactosaminidase as a therapeutic agent for central nervoussystem disorders in Schindler disease and Kawasaki disease; acidicsphingomyelinase (ASM) as a therapeutic agent for central nervous systemdisorders in Niemann-Pick disease; α-galactosidase A as a therapeuticagent for central nervous system disorders in Fabry disease;β-glucuronidase (GUSB) as a therapeutic agent for central nervous systemdisorders in Sly syndrome; heparan N-sulfatase (SGSH),α-N-acetylglucosaminidase (NAGLU), acetyl CoA:α-glucosaminideN-acetyltransferase and N-Acetylglucosamine-6-sulfate sulfatase astherapeutic agents for central nervous system disorders in Sanfilipposyndrome; acid ceramidase (AC) as a therapeutic agent for centralnervous system disorders in Farber disease; amylo-1,6-glucosidase as atherapeutic agent for central nervous system disorders in Cori's disease(Forbes-Cori's disease); sialidase as a therapeutic agent for centralnervous system disorders in sialidase deficiency; palmitoyl proteinthioesterase 1 (PPT-1) as a therapeutic agent for central nervous systemdisorders in neuronal ceroid lipofuscinosis or Santavuori-Haltiadisease; tripeptidyl-peptidase 1 (TPP-1) as a therapeutic agent forcentral nervous system disorders in neuronal ceroid lipofuscinosis orJansky-Bielschowsky disease; hyaluronidase 1 as a therapeutic agent forcentral nervous system disorders in hyaluronidase deficiency; acidicα-glucosidase (GAA) as therapeutic agents for central nervous systemdisorders in Pompe's disease; CLN1 and CLN2 as therapeutic agents forcentral nervous system disorders in Batten disease. In particular, theanti-hTfR antibody of the present invention, after passing through theblood-brain barrier, reaches the brain parenchyma and the hippocampusnerve-like cells of the cerebrum, and Purkinje cells of the cerebellum,and is expected further to reach nerve-like cells of the striatum of thecerebrum and the nerve-like cells of the substantia nigra of themesencephalon. Therefore, the anti-hTfR antibody can be fused withproteins which need to exhibit their functions in those tissues or cellsto strength the pharmacological effects of the proteins. Medicalapplications of it, however, are not limited thereto.

Besides, the one that is used as a therapeutic agent in the presentinvention may be used for preventing the onset of a disease.

Further, examples of proteins that can exhibit their pharmacologicaleffects when linked to the anti-hTfR antibody include: lysosomalenzymes, ciliary neurotrophic factor (CNTF), glial cell line derivedneurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4/5,neurotrophin-6, neuregulin-1, erythropoietin, darbepoetin, activin,basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epidermal growth factor (EGF), vascular endothelial growthfactor (VEGF), interferon α, interferon β, interferon γ, interleukin 6,granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), cytokines, tumor necrosis factor a receptor (TNF-α receptor),PD-1 ligands, PD-L1, PD-L2, enzymes having β-amyloid-degrading activity,anti-β-amyloid antibody, anti-BACE antibody, anti-EGFR antibody,anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-HER2antibody, anti-TNF-α antibody, anti-CTLA-4 antibody, and other antibodymedicines.

Lysosomal enzymes linked to the anti-hTfR antibody can be used as atherapeutic agent for central nervous system disorders in lysosomalstorage diseases; CNTF as a therapeutic agent for amyotrophic lateralsclerosis; GDNF, neurotrophin-3 and neurotrophin-4/5 as therapeuticagents for cerebral ischemia; GDNF as a therapeutic agent forParkinson's disease; neuregulin-1 as a therapeutic agent forschizophrenia; erythropoietin and darbepoetin as therapeutic agents forcerebral ischemia; bFGF and FGF2 as therapeutic agents for traumaticcentral nervous system disorders; for recovery after brain surgery andspinal surgery; enzymes having β-amyloid-degrading activity,anti-β-amyloid antibody and anti-BACE antibody as therapeutic agents forAlzheimer's disease; anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1antibody, anti-PD-L2 antibody, anti-HER2 antibody, and anti-CTLA-4antibody, as therapeutic agents for tumors of central nervous systemincluding brain tumor; and TNFαR-anti-hTfR antibody as therapeuticagents for a cerebral ischemia and encephalitis.

Possible candidates for a “different protein (A)” to be fused to theanti-hTfR antibody generally include those therapeutic agents fordiseases such as neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease, and Huntington's disease; mental disorders such asschizophrenia and depression; multiple sclerosis; amyotrophic lateralsclerosis; tumors of the central nervous system including brain tumor;lysosomal storage diseases accompanied by encephalopathy; glycogenosis;muscular dystrophy; cerebral ischemia; encephalitis; prion diseases;traumatic central nervous system disorders. In addition, therapeuticagent for viral and bacterial central nervous system diseases can alsobe candidates for a different protein (A) to be fused to the anti-hTfRantibody, in general. Further, pharmaceutical agents that can be usedfor recovery after brain surgery or spinal surgery can also becandidates for a “different protein (A)” to be fused to the anti-hTfRantibody, in general.

In addition to the above mentioned natural-type (wild-type) proteins, adifferent protein (A) to be linked to the anti-hTfR antibody may also beone of their analogues in which one or more amino acids of thosenatural-type (wild-type) proteins are modified, e.g., replaced withother amino acids or deleted, insofar as they fully or partly have thefunctions of their respective original proteins. When replacing one ormore amino acids with other amino acids, the number of amino acids to bereplaced is preferably 1-10, more preferably 1-5, still more preferably1-3. When deleting one or more amino acids, the number of amino acids tobe deleted is preferably 1-10, more preferably 1-5, still morepreferably 1-3. A combination of such substitution and deletion of aminoacids can also be carried out to prepare desired analogues. Further,amino acid sequences produced by adding one or more amino acids inside,or on the N-terminal side or on the C-terminal side of, the amino acidsequence of natural-type (wild-type) proteins or their analogues, arealso included in the proteins mentioned above insofar as they fully orpartly have the functions of their respective original proteins. Thenumber of amino acids to be added here is preferably 1-10, morepreferably 1-5, still more preferably 1-3. It is also possible toprepare desired analogues to the original proteins by combiningaddition, substitution, and deletion of amino acids.

Besides, in the case where a mutation is introduced into a differentprotein (A) by adding one or more amino acids on its C-terminus or theN-terminus, if the added amino acids are positioned between the proteinand the anti-hTfR antibody when they are fused, the added amino acidsconstitute part of a linker.

The natural-type human acidic α-glucosidase (hGAA) is a lysosomal enzymecomposed of 883 amino acids sequence set forth as SEQ ID NO: 55.However, one composed of 896 amino acids set forth as SEQ ID NO: 56 inwhich 13 amino acids are further added to the N-terminal side of theamino acid sequence set forth as SEQ ID NO: 55 is also included in thenatural-type hGAA.

An example of specific embodiments of fusion proteins of the presentinvention between the anti-hTfR antibody and a different protein (A) isa type in which the anti-hTfR antibody heavy chain is linked, on theC-terminus thereof and via the amino acid sequence Gly-Ser, as a linkersequence, to the natural-type hGAA. Examples of such a type of fusionproteins include one whose light chain consists of the amino acidsequence set forth as SEQ ID NO: 23, and whose heavy chain consists ofthe amino acid sequence set forth as SEQ ID NO: 66 or 68, and is linked,on the C-terminal side thereof and via the linker sequence Gly-Ser, tohGAA, by peptide bonds. The one having the amino acid sequence set forthas SEQ ID NO: 66 is an IgG1-type anti-hTfR antibody, and the one havingthe amino acid sequence set forth as SEQ ID NO: 68 is an IgG4-typeanti-hTfR antibody. A nucleotide sequence encoding the one having theamino acid sequence set forth as SEQ ID NO: 66 is set forth as, forexample, SEQ ID NO: 67, and a nucleotide sequence encoding the onehaving the amino acid sequence set forth as SEQ ID NO: 68 is set forthas, for example, SEQ ID NO: 69.

The human acidic α-glucosidase (hGAA) is also called α-1,4-glucosidaseor acidic maltase. hGAA has an activity of degrading glycogen byhydrolyzing the α-1,4- and α-1,6-glycoside bonds of the glycogen inlysosome. Pompe's disease, also called glycogen storage disease type II(GSD II), is a disease caused by intracellular glycogen accumulationassociated with deficiency in acidic α-glucosidase (acidic maltase)activity in lysosome. Pompe's disease patients may manifest centralnervous system disorders. hGAA conjugated with the anti-hTfR antibodycan be used as a therapeutic agent for central nervous system disordersaccompanying Pompe's disease.

In the present invention, though the term “human GAA” or “hGAA” refers,in particular, to the hGAA having the same amino acid sequence as thenatural-type hGAA, it also includes those amino acid sequences producedby introducing a mutation, such as substitution, deletion, addition andthe like, into the natural-type hGAA, insofar as they have the hGAAactivity. When replacing one or more of the amino acids of the aminoacid sequence of hGAA with other amino acids, the number of amino acidsto be replaced is preferably 1-10, more preferably 1-5, still morepreferably 1-3, even more preferably 1-2. When deleting one of moreamino acids of the amino acid sequence of hGAA, the number of aminoacids to be deleted is 1-10, more preferably 1-5, still more preferably1-3, and even more preferably 1-2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hGAA, they may be added, inside, or onthe N-terminal side or the C-terminal side of, the amino acid sequenceof hGAA, and the number of amino acids to be added is preferably 1-10,more preferably 1-5, still more preferably 1-3, even more preferably1-2. It is also possible to introduce a combined mutation of suchaddition, substitution, and deletion. The amino acid sequence of themutated hGAA has a homology of preferably not lower than 80%, morepreferably not lower than 90%, still more preferably not lower than 95%to the amino acid sequence of the original hGAA.

The statement that hGAA has the hGAA activity herein means that the hGAAfused to anti-hTfR antibody has an activity not lower than 3% of theactivity that the natural-type hGAA intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hGAAintrinsically has. The same also applies if the hGAA fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and hGAA can beproduced by, for example, transforming host cells, such as mammaliancells, with an expression vector having an incorporated DNA fragmentcomprising the nucleotide sequence set forth as SEQ ID NO: 59 thatencodes the amino acid sequence set forth as SEQ ID NO: 58 and anexpression vector having an incorporated DNA fragment comprising thenucleotide sequence set forth as SEQ ID NO: 24 that encodes theanti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Pompe's disease, inparticular, a therapeutic agent for central nervous system disorders inPompe's disease. The fusion protein obtained in this manner is a fusionprotein of an IgG4-type humanized anti-hTfR antibody and hGAA.

Besides, in the case where the anti-hTfR antibody or hGAA is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and hGAA, the added amino acids constitute part of alinker.

The natural-type human I2S (hI2S) is a lysosomal enzyme composed of 525amino acids sequence set forth as SEQ ID NO:50. A specific example offusion proteins of the present invention between the anti-hTfR antibodyand a different protein (A) is a type in which the anti-hTfR antibodyheavy chain is fused, on the C-terminus thereof and via the amino acidsequence Gly-Ser as a linker sequence, to the natural-type human I2S.Examples of such a type of fusion proteins include one whose light chainconsists of the amino acid sequence set forth as SEQ ID NO: 23, andwhose heavy chain is linked, on the C-terminal side thereof and via alinker consisting of the amino acid sequence Gly-Ser, to human I2S, bypeptide bonds, to form the amino acid sequence set forth as SEQ ID NO:53. The one having the amino acid sequence set forth as SEQ ID NO: 53 isa fusion product of the heavy chain of an IgG1-type anti-hTfR antibodyand hI2S, and the heavy chain moiety has the amino acid sequence setforth as SEQ ID NO: 66. By replacing this heavy chain moiety with theamino acid sequence set forth as SEQ ID NO: 68, it is also possible tofuse an IgG4-type anti-hTfR antibody with hI2S.

The human I2S (hI2S) has an activity of hydrolyzing the sulfate bonds ofheparan sulfate and dermatan sulfate belonging to glycosaminoglycan.Hunter syndrome patients having genetic abnormality in this enzymeresult in symptoms such as skeletal abnormality as partial hydrolysatesof heparan sulfate and dermatan sulfate accumulate in the tissues of theliver, the spleen and the like due to the abnormal metabolism of heparansulfate and dermatan sulfate. Further, Hunter syndrome patients maymanifest central nervous system disorders. hI2S conjugated with theanti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying Hunter syndrome.

In the present invention, though the term “human I2S” or “hI2S” refers,in particular, to the hI2S having the same amino acid sequence as thenatural-type hI2S, it also includes those amino acid sequences producedby introducing a mutation, such as substitution, deletion, addition andthe like, into the amino acid sequence of the natural-type hI2S, insofaras they have the I2S activity. When replacing one or more of the aminoacids of the amino acid sequence of hI2S with other amino acids, thenumber of amino acids to be replaced is preferably 1-10, more preferably1-5, still more preferably 1-3, even more preferably 1-2. When deletingone or more amino acids of the amino acid sequence of hI2S, the numberof amino acids to be deleted is 1-10, more preferably 1-5, still morepreferably 1-3, and even more preferably 1-2. It is also possible tointroduce a combined mutation of such substitution and deletion of aminoacids. When adding one or more amino acids to hI2S, they may be added,inside, or on the N-terminal side or the C-terminal side of, the aminoacid sequence of hI2S, and the number of amino acids to be added ispreferably 1-10, more preferably 1-5, still more preferably 1-3, evenmore preferably 1-2. It is also possible to introduce a combinedmutation of such addition, substitution, and deletion of the amino acid.The amino acid sequence of the mutated hI2S has a homology of preferablynot lower than 80%, more preferably not lower than 90%, still morepreferably not lower than 95% to the amino acid sequence of the originalhI2S.

The statement that hI2S has the I2S activity herein means that the hI2Sfused to anti-hTfR antibody has an activity not lower than 3% of theactivity that the natural-type hI2S intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hI2Sintrinsically has. The same also applies if the hI2S fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and hGAA can beproduced by, for example, transforming host cells, such as mammaliancells, with an expression vector having an incorporated DNA fragmentcomprising the nucleotide sequence set forth as SEQ ID NO:54 thatencodes the amino acid sequence set forth as SEQ ID NO:53 and anexpression vector having an incorporated DNA fragment comprising thenucleotide sequence set forth as SEQ ID NO:24 that encodes the aminoacid sequence set forth as SEQ ID NO:23 (anti-hTfR antibody lightchain), and then culturing the host cells. The fusion protein thusproduced can be used as a therapeutic agent for Pompe's disease, inparticular, a therapeutic agent for central nervous system disordersaccompanying Pompe's disease. The fusion protein obtained in this manneris a fusion protein of an IgG1-type humanized anti-hTfR antibody andhGAA.

Besides, in the case where the anti-hTfR antibody or human I2S ismutated by adding one or more amino acids to them on the C-terminus orthe N-terminus thereof, if the added amino acids are positioned betweenthe anti-hTfR antibody and human I2S, the added amino acids constitutepart of a linker.

The natural-type human α-L-iduronidase (hIDUA) is a lysosomal enzymeconsisting of an amino acid sequence set forth as a SEQ ID NO: 75 or 76.The hIDUA set forth as SEQ ID NO: 76 is a type in which Ala-Pro is addedon the N-terminal side of the amino acid sequence set forth as SEQ IDNO: 75.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hIDUA. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hIDUA by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SE ID NO:66, and an anti-hTfR antibody of IgG4 type has the amino acid sequenceset forth as SEQ ID NO: 68.

The human α-L-iduronidase (hIDUA) is a lysosomal enzyme hydrolyzing aniduronic acid bond present in the dermatan sulfate or heparan sulfatemolecule. The Hurler syndrome, which is also referred to asmucopolysaccharidosis type I, is a disease caused by accumulation ofdermatan sulfate or the like in cells due to deficiency ofα-L-iduronidase activity in the lysosome. Hurler syndrome patients mayhave an accompanying central nervous system disorder. The hIDUA bound tothe anti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying the Hurler syndrome.

In the present invention, though the term “human IDUA” or “hIDUA”refers, in particular, to the hIDUA having the same amino acid sequenceas the natural-type hIDUA, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehIDUA, insofar as they have the hIDUA activity. When replacing one ormore of the amino acids of the amino acid sequence of hIDUA with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hIDUA, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hIDUA, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hIDUA, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hIDUA has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hIDUA.

The statement that hIDUA has the hIDUA activity herein means that thehIDUA fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hIDUA intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hIDUAintrinsically has. The same also applies if the hIDUA fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hIDUA can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 90 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Hurler syndrome, inparticular, a therapeutic agent for central nervous system disordersaccompanying the Hurler syndrome. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hIDUA.

Besides, in the case where the anti-hTfR antibody or hIDUA is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hIDUA, the added amino acids constitute partof a linker.

The natural-type human palmitoyl protein thioesterase 1 (hPPT-1) is atype of the lysosomal enzyme consisting of the amino acid sequence setforth as SEQ ID NO: 77.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hPPT-1. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hPPT-1 by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease)is a disease caused by accumulation of ceroid lipofuscin in cells due todeficiency of palmitoyl protein thioesterase 1 activity in lysosome.Neuronal ceroid lipofuscinosis patients may have an accompanying centralnervous system disorder. The hPPT-1 bound to the anti-hTfR antibody canbe used as a therapeutic agent for central nervous system disordersaccompanying the neuronal ceroid lipofuscinosis.

In the present invention, though the term “human PPT-1” or “hPPT-1”refers, in particular, to the hPPT-1 having the same amino acid sequenceas the natural-type hPPT-1, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehPPT-1, insofar as they have the hPPT-1 activity. When replacing one ormore of the amino acids of the amino acid sequence of hPPT-1 with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hPPT-1, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hPPT-1, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hPPT-1, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hPPT-1 has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hPPT-1.

The statement that hPPT-1 has the hPPT-1 activity herein means that thehPPT-1 fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hPPT-1 intrinsically has. However,the activity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hPPT-1intrinsically has. The same also applies if the hPPT-1 fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hPPT-1 can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 100 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for neuronal ceroidlipofuscinosis, in particular, a therapeutic agent for central nervoussystem disorders accompanying the neuronal ceroid lipofuscinosis. Thefusion protein thus obtained is a fusion protein between the humanizedanti-hTfR antibody of IgG4 type and the hPPT-1.

Besides, in the case where the anti-hTfR antibody or hPPT-1 is mutatedby adding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hPPT-1, the added amino acids constitute partof a linker.

The natural-type human acidic sphingomyelinase (hASM) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 78.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hASM. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hASM by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The human acidic sphingomyelinase (hASM) is a lysosomal enzymehydrolyzing sphingomyelin into choline phosphate and ceramide. TheNiemann-Pick disease is classified into A to F types according to adifference in cause and condition of the disease. Among them, types Aand B are caused by genetic deficiency of acidic sphingomyelinase (ASM).The Niemann-Pick disease is a disease caused by accumulation ofsphingomyelin in cells due to deficiency of acidic sphingomyelinaseactivity in lysosome. Niemann-Pick disease patients may have anaccompanying central nervous system disorder. The hASM bound to theanti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying the Niemann-Pick disease.

In the present invention, though the term “human ASM” or “hASM” refers,in particular, to the hASM having the same amino acid sequence as thenatural-type hASM, it also includes those amino acid sequences producedby introducing a mutation, such as substitution, deletion, addition andthe like, into the amino acid sequence of the natural-type hASM, insofaras they have the hASM activity. When replacing one or more of the aminoacids of the amino acid sequence of hASM with other amino acids, thenumber of amino acids to be replaced is preferably 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. When deleting one or more amino acids of the amino acid sequenceof hASM, the number of amino acids to be deleted is 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. It is also possible to introduce a combined mutation of suchsubstitution and deletion of amino acids. When adding one or more aminoacids to hASM, they may be added, inside, or on the N-terminal side orthe C-terminal side of, the amino acid sequence of hASM, and the numberof amino acids to be added is preferably 1 to 10, more preferably 1 to5, still more preferably 1 to 3, even more preferably 1 or 2. It is alsopossible to introduce a combined mutation of such addition,substitution, and deletion of the amino acid. The amino acid sequence ofthe mutated hASM has a homology of preferably not lower than 80%, morepreferably not lower than 90%, still more preferably not lower than 95%to the amino acid sequence of the original hASM.

The statement that hASM has the hASM activity herein means that the hASMfused to anti-hTfR antibody has an activity not lower than 3% of theactivity that the natural-type hASM intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hASMintrinsically has. The same also applies if the hASM fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hASM can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 106 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Niemann-Pickdisease, in particular, a therapeutic agent for central nervous systemdisorders accompanying the Niemann-Pick disease. The fusion protein thusobtained is a fusion protein between the humanized anti-hTfR antibody ofIgG4 type and the hASM.

Besides, in the case where the anti-hTfR antibody or hASM is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hASM, the added amino acids constitute partof a linker.

The natural-type human arylsulfatase A (hARSA) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 79.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hARSA. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hARSA by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The metachromatic white matter degeneration (metachromaticleukodystrophy) is a disease caused by accumulation of sulfatide or thelike in cells due to deficiency of arylsulfatase A activity in lysosome.Metachromatic white matter degeneration patients may have anaccompanying central nervous system disorder. The hARSA bound to theanti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying the metachromatic white matterdegeneration.

In the present invention, though the term “human ARSA” or “hARSA”refers, in particular, to the hARSA having the same amino acid sequenceas the natural-type hARSA, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehARSA, insofar as they have the hARSA activity. When replacing one ormore of the amino acids of the amino acid sequence of hARSA with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hARSA, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hARSA, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hARSA, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hARSA has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hARSA.

The statement that hARSA has the hARSA activity herein means that thehARSA fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hARSA intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hARSAintrinsically has. The same also applies if the hARSA fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hARSA can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 115 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for metachromatic whitematter degeneration, in particular, a therapeutic agent for centralnervous system disorders accompanying the metachromatic white matterdegeneration. The fusion protein thus obtained is a fusion proteinbetween the humanized anti-hTfR antibody of IgG4 type and the hARSA.

Besides, in the case where the anti-hTfR antibody or hARSA is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hARSA, the added amino acids constitute partof a linker.

The natural-type human heparan N-sulfatase (hSGSH) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 80.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hSGSH. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hSGSH by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The Sanfilippo syndrome, which is also referred to asmucopolysaccharidosis type II (MPS type III), is a disease caused byaccumulation of heparan sulfate in cells due to deficiency of heparanN-sulfatase activity in lysosome. It is to be noted that the Sanfilipposyndrome may be caused by deficiency of other enzymes such asα-N-acetylglucosaminidase. Sanfilippo syndrome patients may have anaccompanying central nervous system disorder. The hSGSH bound to theanti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying the Sanfilippo syndrome.

In the present invention, though the term “human SGSH” or “hSGSH”refers, in particular, to the hSGSH having the same amino acid sequenceas the natural-type hSGSH, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehSGSH, insofar as they have the hSGSH activity. When replacing one ormore of the amino acids of the amino acid sequence of hSGSH with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hSGSH, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hSGSH, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hSGSH, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hSGSH has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hSGSH.

The statement that hSGSH has the hSGSH activity herein means that thehSGSH fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hSGSH intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hSGSHintrinsically has. The same also applies if the hSGSH fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hSGSH can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 124 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Sanfilipposyndrome, in particular, a therapeutic agent for central nervous systemdisorders accompanying the Sanfilippo syndrome. The fusion protein thusobtained is a fusion protein between the humanized anti-hTfR antibody ofIgG4 type and the hSGSH.

Besides, in the case where the anti-hTfR antibody or hSGSH is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hSGSH, the added amino acids constitute partof a linker.

The natural-type human glucocerebrosidase (hGBA) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 81.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hGBA. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hGBA by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The human glucocerebrosidase (hGBA) is a lysosomal enzyme hydrolyzingglycolipid glucocerebroside (glucosylceramide). The Gaucher disease is adisease caused by accumulation of glucocerebroside in cells due todeficiency of glucocerebrosidase activity in lysosome. Gaucher diseasepatients may have an accompanying central nervous system disorder. ThehGBA bound to the anti-hTfR antibody can be used as a therapeutic agentfor central nervous system disorders accompanying the Gaucher disease.

In the present invention, though the term “human GBA” or “hGBA” refers,in particular, to the hGBA having the same amino acid sequence as thenatural-type hGBA, it also includes those amino acid sequences producedby introducing a mutation, such as substitution, deletion, addition andthe like, into the amino acid sequence of the natural-type hGBA, insofaras they have the hGBA activity. When replacing one or more of the aminoacids of the amino acid sequence of hGBA with other amino acids, thenumber of amino acids to be replaced is preferably 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. When deleting one or more amino acids of the amino acid sequenceof hGBA, the number of amino acids to be deleted is preferably 1 to 10,more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. It is also possible to introduce a combined mutationof such substitution and deletion of amino acids. When adding one ormore amino acids to hGBA, they may be added, inside, or on theN-terminal side or the C-terminal side of, the amino acid sequence ofhGBA, and the number of amino acids to be added is preferably 1 to 10,more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. It is also possible to introduce a combined mutationof such addition, substitution, and deletion of the amino acid. Theamino acid sequence of the mutated hGBA has a homology of preferably notlower than 80%, more preferably not lower than 90%, still morepreferably not lower than 95% to the amino acid sequence of the originalhGBA.

The statement that hGBA has the hGBA activity herein means that the hGBAfused to anti-hTfR antibody has an activity not lower than 3% of theactivity that the natural-type hGBA intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hGBAintrinsically has. The same also applies if the hGBA fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hGBA can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 130 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Gaucher disease, inparticular, a therapeutic agent for central nervous system disordersaccompanying the Gaucher disease. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hGBA.

Besides, in the case where the anti-hTfR antibody or hGBA is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hGBA, the added amino acids constitute partof a linker.

The natural-type human tripeptidyl peptidase-1 (hTPP-1) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 82.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hTPP-1. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hTPP-1 by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SE ID NO:66, and an anti-hTfR antibody of IgG4 type has the amino acid sequenceset forth as SEQ ID NO: 68.

The neuronal ceroid lipofuscinosis (including Santavuori-Haltia disease)is a disease caused by accumulation of lipofuscin in cells due todeficiency of tripeptidyl peptidase 1 activity in liposome. Neuronalceroid lipofuscinosis patients may have an accompanying central nervoussystem disorder. The hTPP-1 bound to the anti-hTfR antibody can be usedas a therapeutic agent for central nervous system disorders accompanyingthe neuronal ceroid lipofuscinosis.

In the present invention, though the term “human TPP-1” or “hTPP-1”refers, in particular, to the hTPP-1 having the same amino acid sequenceas the natural-type hTPP-1, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehTPP-1, insofar as they have the hTPP-1 activity. When replacing one ormore of the amino acids of the amino acid sequence of hTPP-1 with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hTPP-1, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hTPP-1, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hTPP-1, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hTPP-1 has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hTPP-1.

The statement that hTPP-1 has the hTPP-1 activity herein means that thehTPP-1 fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hTPP-1 intrinsically has. However,the activity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hTPP-1intrinsically has. The same also applies if the hTPP-1 fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hTPP-1 can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 136 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for neuronal ceroidlipofuscinosis, in particular, a therapeutic agent for central nervoussystem disorders accompanying the neuronal ceroid lipofuscinosis. Thefusion protein thus obtained is a fusion protein between the humanizedanti-hTfR antibody of IgG4 type and the hTPP-1.

Besides, in the case where the anti-hTfR antibody or hTPP-1 is mutatedby adding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hTPP-1, the added amino acids constitute partof a linker.

The natural-type human α-N-acetylglucosaminidase (hNAGLU) is a type ofthe lysosomal enzyme consisting of the amino acid sequence set forth asSEQ ID NO: 83.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hNAGLU. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hNAGLU by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The Sanfilippo syndrome is a disease caused by accumulation of heparansulfate in cells due to deficiency of α-N-acetylglucosaminidase activityin lysosome. Sanfilippo syndrome patients may have an accompanyingcentral nervous system disorder. The hNAGLU bound to the anti-hTfRantibody can be used as a therapeutic agent for central nervous systemdisorders accompanying the Sanfilippo syndrome.

In the present invention, though the term “human NAGLU” or “hNAGLU”refers, in particular, to the hNAGLU having the same amino acid sequenceas the natural-type hNAGLU, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehNAGLU, insofar as they have the hNAGLU activity. When replacing one ormore of the amino acids of the amino acid sequence of hNAGLU with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hNAGLU, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hNAGLU, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hNAGLU, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hNAGLU has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hNAGLU.

The statement that hNAGLU has the hNAGLU activity herein means that thehNAGLU fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hNAGLU intrinsically has. However,the activity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hNAGLUintrinsically has. The same also applies if the hNAGLU fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hNAGLU can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 142 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Sanfilipposyndrome, in particular, a therapeutic agent for central nervous systemdisorders accompanying the Sanfilippo syndrome. The fusion protein thusobtained is a fusion protein between the humanized anti-hTfR antibody ofIgG4 type and the hNAGLU.

Besides, in the case where the anti-hTfR antibody or hNAGLU is mutatedby adding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hNAGLU, the added amino acids constitute partof a linker.

The natural-type human β-glucuronidase (hGUSB) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 84.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hGUSB. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hGUSB by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The Sly syndrome, which is also referred to as mucopolysaccharidosistype VII (MPS type VII), is a disease caused by accumulation ofmucopolysaccharides in cells due to deficiency of β-glucuronidaseactivity in lysosome. Sly syndrome patients may have an accompanyingcentral nervous system disorder. The hGUSB bound to the anti-hTfRantibody can be used as a therapeutic agent for central nervous systemdisorders accompanying the Sly syndrome.

In the present invention, though the term “human GUSB” or “hGUSB”refers, in particular, to the hGUSB having the same amino acid sequenceas the natural-type hGUSB, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehGUSB, insofar as they have the hGUSB activity. When replacing one ormore of the amino acids of the amino acid sequence of hGUSB with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hGUSB, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hGUSB, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hGUSB, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hGUSB has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hGUSB.

The statement that hGUSB has the hGUSB activity herein means that thehGUSB fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hGUSB intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hGUSBintrinsically has. The same also applies if the hGUSB fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hGUSB can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 150 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus obtained is a fusion protein between the humanized anti-hTfRantibody of IgG4 type and the hGUSB. The fusion protein thus producedcan be used as a therapeutic agent for Sly syndrome, in particular, atherapeutic agent for central nervous system disorders accompanying theSly syndrome.

Besides, in the case where the anti-hTfR antibody or hGUSB is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hGUSB, the added amino acids constitute partof a linker.

The natural-type human galactosylceramidase (hGALC) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 85.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hGALC. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hGALC by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The Krabbe disease is a disease caused by deficiency ofgalactosylceramidase activity. Krabbe disease patients may have anaccompanying central nervous system disorder. The hGALC bound to theanti-hTfR antibody can be used as a therapeutic agent for centralnervous system disorders accompanying the Krabbe disease.

In the present invention, though the term “human GALC” or “hGALC”refers, in particular, to the hGALC having the same amino acid sequenceas the natural-type hGALC, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehGALC, insofar as they have the hGALC activity. When replacing one ormore of the amino acids of the amino acid sequence of hGALC with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hGALC, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hGALC, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hGALC, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hGALC has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hGALC.

The statement that hGALC has the hGALC activity herein means that thehGALC fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hGALC intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hGALCintrinsically has. The same also applies if the hGALC fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hGALC can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 158 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Krabbe disease, inparticular, a therapeutic agent for central nervous system disordersaccompanying the Krabbe disease. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hGALC.

Besides, in the case where the anti-hTfR antibody or hGALC is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hGALC, the added amino acids constitute partof a linker.

The natural-type human acidic ceramidase (hAC) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 86.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hAC. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hAC by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The Farber disease is a disease caused by accumulation of ceramide incells due to deficiency of acidic ceramidase activity in lysosome.Farber disease patients may have an accompanying central nervous systemdisorder. The hAC bound to the anti-hTfR antibody can be used as atherapeutic agent for central nervous system disorders accompanying theFarber disease.

In the present invention, though the term “human AC” or “hAC” refers, inparticular, to the hAC having the same amino acid sequence as thenatural-type hAC, it also includes those amino acid sequences producedby introducing a mutation, such as substitution, deletion, addition andthe like, into the amino acid sequence of the natural-type hAC, insofaras they have the hAC activity. When replacing one or more of the aminoacids of the amino acid sequence of hAC with other amino acids, thenumber of amino acids to be replaced is preferably 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, even more preferably 1or 2. When deleting one or more amino acids of the amino acid sequenceof hAC, the number of amino acids to be deleted is preferably 1 to 10,more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. It is also possible to introduce a combined mutationof such substitution and deletion of amino acids. When adding one ormore amino acids to hAC, they may be added, inside, or on the N-terminalside or the C-terminal side of, the amino acid sequence of hAC, and thenumber of amino acids to be added is preferably 1 to 10, more preferably1 to 5, still more preferably 1 to 3, even more preferably 1 or 2. It isalso possible to introduce a combined mutation of such addition,substitution, and deletion of the amino acid. The amino acid sequence ofthe mutated hAC has a homology of preferably not lower than 80%, morepreferably not lower than 90%, still more preferably not lower than 95%to the amino acid sequence of the original hAC.

The statement that hAC has the hAC activity herein means that the hACfused to anti-hTfR antibody has an activity not lower than 3% of theactivity that the natural-type hAC intrinsically has. However, theactivity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hACintrinsically has. The same also applies if the hAC fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hAC can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 166 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for Farber disease, inparticular, a therapeutic agent for central nervous system disordersaccompanying the Farber disease. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hAC.

Besides, in the case where the anti-hTfR antibody or hAC is mutated byadding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hAC, the added amino acids constitute part ofa linker.

The natural-type human α-L-fucosidase (hFUCA1) is a type of thelysosomal enzyme consisting of the amino acid sequence set forth as SEQID NO: 87.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hFUCAl. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hFUCA1 by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The fucosidosis is a disease caused by accumulation of fucose-containingoligosaccharides in cells due to deficiency of α-L-fucosidase activityin lysosome. Fucosidosis patients may have an accompanying centralnervous system disorder. The hFUCA1 bound to the anti-hTfR antibody canbe used as a therapeutic agent for central nervous system disordersaccompanying the fucosidosis.

In the present invention, though the term “human FUCA1” or “hFUCA1”refers, in particular, to the hFUCA1 having the same amino acid sequenceas the natural-type hFUCA1, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehFUCA1, insofar as they have the hFUCA1 activity. When replacing one ormore of the amino acids of the amino acid sequence of hFUCA1 with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hFUCA1, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hFUCA1, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hFUCA1, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hFUCA1 has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hFUCA1.

The statement that hFUCA1 has the hFUCA1 activity herein means that thehFUCA1 fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hFUCA1 intrinsically has. However,the activity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hFUCA1intrinsically has. The same also applies if the hFUCA1 fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hFUCA1 can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 174 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for fucosidosis, inparticular, a therapeutic agent for central nervous system disordersaccompanying the fucosidosis. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hFUCA1.

Besides, in the case where the anti-hTfR antibody or hFUCA1 is mutatedby adding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hFUCA1, the added amino acids constitute partof a linker.

The natural-type human α-mannosidase (hLAMAN) is a type of the lysosomalenzyme consisting of the amino acid sequence set forth as SEQ ID NO: 88.

A specific example of the fusion protein between the anti-hTfR antibodyand a different protein (A) in the present invention is a type in whichthe anti-hTfR antibody heavy chain is fused, on the C-terminus thereofand via the amino acid sequence Gly-Ser as a linker sequence, to thenatural-type hLAMAN. Examples of such fusion proteins include one whoselight chain consists of the amino acid sequence set forth as SEQ ID NO:23 and whose heavy chain consists of the amino acid sequence set forthas SEQ ID NO: 66 or 68, and is bound, on the C-terminal side and via thelinker sequence Gly-Ser, to the hLAMAN by a peptide bond. An anti-hTfRantibody of IgG1 type has the amino acid sequence set forth as SEQ IDNO: 66, and an anti-hTfR antibody of IgG4 type has the amino acidsequence set forth as SEQ ID NO: 68.

The human α-mannosidase (hLAMAN) is a lysosomal enzyme hydrolyzingα-type mannose. The α-mannosidosis is a disease caused by accumulationof mannose-containing oligosaccharides in cells due to deficiency ofα-mannosidase activity in lysosome. α-Mannosidosis patents may have anaccompanying central nervous disorder. The hLAMAN bound to the anti-hTfRantibody can be used as a therapeutic agent for central nervousdisorders accompanying the α-mannosidosis.

In the present invention, though the term “human LAMAN” or “hLAMAN”refers, in particular, to the hLAMAN having the same amino acid sequenceas the natural-type hLAMAN, it also includes those amino acid sequencesproduced by introducing a mutation, such as substitution, deletion,addition and the like, into the amino acid sequence of the natural-typehLAMAN, insofar as they have the hLAMAN activity. When replacing one ormore of the amino acids of the amino acid sequence of hLAMAN with otheramino acids, the number of amino acids to be replaced is preferably 1 to10, more preferably 1 to 5, still more preferably 1 to 3, even morepreferably 1 or 2. When deleting one or more amino acids of the aminoacid sequence of hLAMAN, the number of amino acids to be deleted ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such substitution and deletion of amino acids. Whenadding one or more amino acids to hLAMAN, they may be added, inside, oron the N-terminal side or the C-terminal side of, the amino acidsequence of hLAMAN, and the number of amino acids to be added ispreferably 1 to 10, more preferably 1 to 5, still more preferably 1 to3, even more preferably 1 or 2. It is also possible to introduce acombined mutation of such addition, substitution, and deletion of theamino acid. The amino acid sequence of the mutated hLAMAN has a homologyof preferably not lower than 80%, more preferably not lower than 90%,still more preferably not lower than 95% to the amino acid sequence ofthe original hLAMAN.

The statement that hLAMAN has the hLAMAN activity herein means that thehLAMAN fused to anti-hTfR antibody has an activity not lower than 3% ofthe activity that the natural-type hLAMAN intrinsically has. However,the activity is preferably not lower than 10%, more preferably not lowerthan 20%, still more preferably not lower than 50%, even more preferablynot lower than 80% of the activity that the natural-type hLAMANintrinsically has. The same also applies if the hLAMAN fused to theanti-hTfR antibody is mutated.

The fusion protein between the anti-hTfR antibody and the hLAMAN can beproduced by, for example, transforming host cells such as mammaliancells, with an expression vector having an incorporated DNA segmentencoding the amino acid sequence set forth as SEQ ID NO: 182 and anexpression vector having an incorporated DNA segment encoding ananti-hTfR antibody light chain having the amino acid sequence set forthas SEQ ID NO: 23, and then culturing the host cells. The fusion proteinthus produced can be used as a therapeutic agent for α-mannosidosis, inparticular, a therapeutic agent for central nervous system disordersaccompanying the α-mannosidosis. The fusion protein thus obtained is afusion protein between the humanized anti-hTfR antibody of IgG4 type andthe hLAMAN.

Besides, in the case where the anti-hTfR antibody or hLAMAN is mutatedby adding one or more amino acids to them on the C-terminus or theN-terminus thereof, if the added amino acids are positioned between theanti-hTfR antibody and the hLAMAN, the added amino acids constitute partof a linker.

As examples of fusion proteins between the anti-hTfR antibody and adifferent protein (A), fusion proteins having hGAA, hI2S, hIDUA, hPPT-1,hASM, hARSA, hSGSH, hGBA, hTTP-1, hNAGLU, hGUSB, hAC, hFUcal and hLAMAN,respectively, as the different protein (A) are described above, butthere is no particular limitation as to the amino acid sequences of theCDRs of the anti-hTfR antibody heavy chain and light chain in preferredembodiments of fusion proteins between the anti-hTfR antibody and such adifferent protein (A) insofar as the antibody has a specific affinity tohTfR.

However, anti-TfR antibodies used herein are those whose dissociationconstant is as follows, in particular, as measured by the methoddescribed in Example 7:

dissociation constant with human TfR: preferably not greater than1×10⁻¹⁰ M, more preferably not greater than 1×10⁻¹¹ M, still morepreferably not greater than 5×10⁻¹² M, even more preferably not greaterthan 1×10⁻¹² M; and

dissociation constant with monkey TfR: preferably not greater than1×10⁻⁹ M, more preferably not greater than 5×10⁻¹⁰ M, still morepreferably not greater than 1×10⁻¹⁰ M, for example not greater than7.5×10⁻¹¹ M.

For example, the dissociation constants with human TfR and monkey TfRare not greater than 1×10⁻¹⁰ M and not greater than 1×10⁻⁹ M, notgreater than 1×10⁻¹¹ M and not greater than 5×10⁻¹⁰ M, not greater than5×10⁻¹² M and not greater than 1×10⁻¹⁰ M, not greater than 5×10⁻¹² M andnot greater than 7.5×10⁻¹¹ M, not greater than 1×10⁻¹² M and not greaterthan 1×10⁻¹⁰ M, or not greater than 1×10⁻¹² M and not greater than7.5×10⁻¹¹ M, respectively. In this context, although there is noparticularly definite lower limit on the dissociation constant withhuman TfR, it can be, for example, 5×10⁻¹³ M or 1×10⁻¹³ M. Althoughthere is no particularly definite lower limit on the dissociationconstant with monkey TfR, it can be, for example, 1×10⁻¹¹ M or 1×10⁻¹²M. The same also applies if the antibody is a single-chain antibody.

It is also possible to link a relatively short peptide chain to theanti-hTfR antibody, in the same manner as in linking a different protein(A) to the anti-hTfR antibody. There is no particular limitation as to apeptide chain to be linked to the anti-hTfR antibody, insofar as thepeptide chain has a desired physiological activity. For example, thereare peptide chains comprising the amino acid sequence of such a regionof various proteins that exhibits a physiological activity. Though thereis no particular limitation as to the length of the peptide chain, theyare composed of preferably 2-200 amino acids, for example of 5-50 aminoacids.

In linking a low-molecular-weight compound to the anti-hTfR antibody,there is no particular limitation as to candidate low-molecular-weightcompounds, but they are such low-molecular-weight compound that thoughneeded to get inside the brain and function there, due to theirinability to pass through the blood-brain barrier as it is, cannot beexpected to function in the brain if simply administered intravenously.Examples of such low-molecular-weight compounds include anticancer drugsuch as cyclophosphamide, ifosfamide, melphalan, busulfan, thioTEPA,nimustine, ranimustine, dacarbazine, procarbazine, temozolomide,carmustine, streptozocin, bendamustine, cisplatin, carboplatin,oxaliplatin, nedaplatin, 5-fluorouracil, sulfadiazine, sulfamethoxazole,methotrexate, trimethoprim, pyrimethamine, fluorouracil, flucytosine,azathioprine, pentostatin, hydroxyurea, fludarabine, cytarabine,gemcitabine, irinotecan, doxorubicin, etoposide, levofloxacin,ciprofloxacin, vinblastine, vincristine, paclitaxel, docetaxel,Mitomycin C, doxorubicin, epirubicin. Further examples oflow-molecular-weight compound to be linked to the anti-hTfR antibodyinclude siRNAs, antisense DNAs, and short peptides.

In linking between the anti-hTfR antibody and a low-molecular-weightcompound, either a low-molecular-weight compound may be linked only toone of the light chain and heavy chain, or it may be linked to both thelight chain and the heavy chain, respectively. Further, insofar as ithas an affinity to hTfR, the anti-hTfR antibody may comprise an aminoacid sequence comprising the whole of part of the light chain variableregion and/or an amino acid sequence comprising the whole of part of theheavy chain variable region.

Candidates for low-molecular-weight compounds to be fused with theanti-hTfR antibody can generally be those therapeutic agents fordiseases such as neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease, Huntington's disease; mental disorders such asschizophrenia, depression; multiple sclerosis; amyotrophic lateralsclerosis; tumor of the central nervous system including brain tumor;lysosomal storage diseases with accompanying encephalopathy;glycogenosis; muscular dystrophy; cerebral ischemia; encephalitis; priondiseases; traumatic disorders of the central nervous system. Further,therapeutic agents for viral and bacterial central nervous systemdiseases can also be candidates, in general, for low-molecular-weightcompounds to be fused with the anti-hTfR antibody. Still further, thosepharmaceutical agents which can be used for recovery after brain surgeryor spinal surgery can also be candidates, in general, forlow-molecular-weight compounds to be fused.

If an anti-hTfR antibody originates from a non-human animal, itsadministration to human could entail a substantial risk of causing anantigen-antibody interaction, thereby provoking adverse side-effects. Byconverting them to humanized antibodies, the antigenicity of non-humananimal antibodies can be reduced and therefore the provocation ofside-effects due to antigen-antibody interaction can be suppressed whenadministered to a human. Further, it has been reported that according toexperiments using monkeys, humanized antibodies are more stable thanmouse antibodies in the blood, and it is expected that their therapeuticeffect can therefore become longer-lasting accordingly. Provocation ofside-effects due to an antigen-antibody interaction can be suppressedalso by employing a human antibody as the anti-hTfR antibody.

A detailed explanation will be given below regarding the case where theanti-hTfR antibody is a humanized antibody or human antibody. In humanantibody light chain, there are λ and κ chains. The light chainconstituting the human antibody may either be λ and κ chain. And inhuman heavy chain, there are γ, μ, α, σ, and ε chains, which correspondto IgG, IgM, IgA, IgD and IgE, respectively. Though the heavy chainconstituting the anti-hTfR antibody may be any of γ, μ, α, σ, and εchains, preferred is a γ chain. Further, in γ chain of human heavychain, there are γ1, γ2, γ3 and γ4 chains, which correspond to IgG1,IgG2, IgG3 and IgG4, respectively. Where the heavy chain constitutingthe anti-hTfR antibody is a γ chain, though the γ chain may be any ofγ1, γ2, γ3 and γ4 chains, preferred is a γ1 or γ4 chain. In the casewhere the anti-hTfR antibody is a humanized antibody or human antibodyand IgG, the human antibody light chain may either be λ chain or κchain, and though the human antibody heavy chain may either be γ1, γ2,γ3 and γ4 chains, preferred is a γ1 or γ4 chain. For example, apreferable embodiment of anti-hTfR antibody includes one whose lightchain is a λ chain and heavy chain is a γ1 chain.

In the case where the anti-hTfR antibody is a humanized antibody or ahuman antibody, the anti-hTfR antibody and a different protein (A) maybe bound together by linking the anti-hTfR antibody, at the N-terminus(or the C-terminus) of the heavy chain or light chain, via a linkersequence or directly, to the C-terminus (or the N-terminus),respectively, of the different protein (A), by peptide bonds. Whenlinking the different protein (A) to the anti-hTfR antibody heavy chainon the N-terminal side (or to the C-terminal side) thereof, theC-terminus (or the N-terminus), respectively, of the different protein(A) is linked to the N-terminus (or the C-terminus) of the γ, μ, α, σ orε chain of anti-hTfR antibody, via a linker sequence or directly, bypeptide bonds. When linking the different protein (A) to the anti-hTfRantibody light chain on the N-terminal side (or the C-terminal side)thereof, the C-terminus (or the N-terminus), respectively, of thedifferent protein (A) in linked to the N-terminus (or the C-terminus) ofthe λ chain and κ chain of anti-hTfR antibody, via a linker sequence ordirectly, by peptide bonds. However, in the case where the anti-hTfRantibody consists of the Fab region, or of the Fab region and the wholeor part of the hinge region (Fab, F(ab′)₂, and F(ab′)), the differentprotein (A) may be linked at the C-terminus (or the N-terminus) thereofand via a linker sequence or directly, to the N-terminus (or theC-terminus), respectively, of the heavy chain or light chain thatconstitutes the Fab, F(ab′)₂ and F(ab′), by peptide bonds.

In a fusion protein produced by linking the different protein (A) to thelight chain of the anti-hTfR antibody which is a humanized antibody, ora human antibody, on the C-terminal side or the N-terminal side thereof,the anti-human transferrin receptor antibody comprises an amino acidsequence comprising the whole or part of the light chain variable regionand the amino acid sequence comprising the whole or part of the heavychain variable region. The anti-hTfR antibody light chain and thedifferent protein (A) here may be linked directly or via a linker.

In a fusion protein produced by linking the different protein (A) to theheavy chain of the anti-hTfR antibody which is a humanized antibody, orhuman antibody, on the C-terminal side or the N-terminal side thereof,the anti-human transferrin receptor antibody comprises an amino acidsequence comprising the whole or part of the light chain variable regionand the amino acid sequence comprising the whole or part of the heavychain variable region. The anti-hTfR antibody heavy chain and thedifferent protein (A) here may be linked directly or via a linker.

When placing a linker sequence between the anti-hTfR antibody or humanantibody and a different protein (A), the linker sequence is preferablya peptide chain consisting of 1-50 amino acids, though the number of theamino acids constituting such a linker sequence may be adjusted asdesired in accordance with the different protein (A) to be linked to theanti-hTfR antibody, like 1-17, 1-10, 10-40, 20-34, 23-31, 25-29, and soon. While there is no particular limitation as to the specific aminoacid sequence of such a linker sequence insofar as the anti-hTfRantibody and the different protein (A) linked by the linker sequenceretain their respective functions (affinity to hTfR, and activity orfunction under a physiological condition), it is preferably composed ofglycine or serine, for example, one consisting of a single amino acideither glycine or serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Ser(SEQ ID NO:3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ IDNO:4), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO:5), or asequence which includes 1-10 or 2-5 of any of those amino acid sequencesconsecutively linked. For example, a linker sequence comprising 27 aminoacids is preferably used that is composed of the amino acid sequenceGly-Ser followed by consecutively linked five copies of the amino acidsequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3). Further, a linker sequencecomprising 25 amino acids is also preferably used that is composed ofconsecutively linked five copies of the amino acid sequenceGly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

Besides, when stated here that a different protein (A) fused with theanti-hTfR antibody or human antibody retains its activity or functionunder a physiological condition, or simply, it “retains the activity”,it means that in comparison with the intrinsic activity of thenatural-type of the different protein (A), not lower than 3% of theactivity or function is retained. However, such an activity or functionis preferably not lower than 10%, more preferably not lower than 20%,still more preferably not lower than 50%, and even more preferably notlower than 80%, in comparison with the intrinsic activity of thenatural-type of the different protein (A). The same also applies wherethe different protein (A) fused with the anti-hTfR antibody is a mutatedone.

A further example of specific embodiments of the fusion protein betweena humanized anti-hTfR antibody or human antibody and a different protein(A) of the present invention is one produced by fusing the anti-hTfRantibody heavy chain, on the C-terminal side thereof, with a differentprotein (A), via a linker sequence consisting of 27 amino acids that iscomposed of the amino acid sequence Gly-Ser followed by consecutivelylinked five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQID NO:3).

In the case where the anti-hTfR antibody is Fab, an example of specificembodiments of fusion proteins between a humanized anti-hTfR antibody orhuman antibody of the present invention and a different protein (A) isone produced by fusing a different protein (A), on the C-terminal sidethereof via a linker sequences, with the region consisting of anti-hTfRantibody heavy chain variable region and its accompanying C_(H)1 region,wherein the linker sequence consists of 25 amino acids that is composedof consecutively linked five copies of the amino acid sequenceGly-Gly-Gly-Gly-Ser (SEQ ID NO:3). Though it is also allowed here thatpart of the hinge region is also besides the C_(H)1 region, the hingeregion does not contain a cysteine residue that would form a disulfidebond between heavy chains.

The specific affinity of the anti-hTfR antibody to hTfR resides mainlyin the amino acid sequences of CDRs of the heavy chain and light chainof the anti-hTfR antibody. There is no particular limitation as to theamino acid sequences of those CDRs insofar as the anti-hTfR antibody hasa specific affinity to monkey hTfR in addition to hTfR.

However, in the present invention, a humanized anti-hTfR antibody or ahuman antibody that is an antibody having a relatively high affinity tohTfR and has affinity both to human and monkey TfRs, simultaneously, isone

whose dissociation constant with human TfR is preferably not greaterthan 1×10⁻¹⁰ M, more preferably not greater than 2.5×10⁻¹¹ M, still morepreferably not greater than 5×10⁻¹² M, and even more preferably notgreater than 1×10⁻¹² M, and

whose dissociation constant with monkey TfR is preferably not greaterthan 1×10⁻⁹ M, more preferably not greater than 5×10⁻¹⁰ M, and stillmore preferably not greater than 1×10⁻¹⁰ M, for example, not greaterthan 7.5×10⁻¹¹ M,

in particular, as measured by the method described in Example 7.

For example, the dissociation constants with human TfR and monkey TfRare not greater than 1×10⁻¹⁰ M and not greater than 1×10⁻⁹ M, notgreater than 1×10⁻¹¹ M and not greater than 5×10⁻¹⁰ M, not greater than5×10⁻¹² M and not greater than 1×10⁻¹⁰ M, not greater than 5×10⁻¹² M andnot greater than 7.5×10⁻¹¹ M, not greater than 1×10⁻¹² M and not greaterthan 1×10⁻¹⁰ M, or not greater than 1×10⁻¹² M and not greater than7.5×10⁻¹¹ M, respectively. In this context, although there is noparticularly definite lower limit on the dissociation constant withhuman TfR, it can be, for example, 5×10⁻¹³ M or 1×10⁻¹³ M. Further,Although there is no particularly definite lower limit on thedissociation constant with monkey TfR, it can be, for example, 1×10⁻¹¹ Mor 1×10⁻¹² M and the like. The same also applies if the antibody is asingle-chain antibody.

Examples of preferable embodiments of the antibody having affinity tohTfR include antibodies in which in the heavy chain variable region,

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 orSEQ ID NO: 63,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 orSEQ ID NO: 14, and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 orSEQ ID NO: 16.

Examples of more specific embodiments of the antibody having affinity tohTfR include antibodies in which in the heavy chain variable region,

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13,and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15.

In the above mentioned preferable embodiments of the antibody havingaffinity to hTfR, and more specific embodiments of the antibody havingaffinity to hTfR, preferred examples of the amino acid sequence of theframework region 3 of the antibody heavy chain include those comprisingthe amino acid sequence set forth as SEQ ID NO: 64.

Examples of preferable combinations of the light chain and heavy chainof the antibody having affinity to hTfR include those having the aminoacid sequences below in the variable regions: a combination of

a light chain in which

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 6 orSEQ ID NO: 7,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 8 orSEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser, and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 10,and

a heavy chain in which

(d) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 orSEQ ID NO: 63,

(e) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 orSEQ ID NO: 14, and

(f) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 orSEQ ID NO: 16.

Examples of specific embodiments of combinations of the light chain andheavy chain of the antibody having affinity to hTfR include those havingthe amino acid sequences below in the variable regions: a combination of

a light chain comprising the amino acid sequences set forth as SEQ IDNO: 6 as CDR1, SEQ ID NO: 8 as CDR2, and SEQ ID NO: 10 as CDR3,respectively, and a heavy chain comprising the amino acid sequences setforth as SEQ ID NO: 62 as CDR1, SEQ ID NO: 13 as CDR2, and SEQ ID NO: 15as CDR3, respectively.

In the above mentioned preferable combinations of the light chain andheavy chain of the antibody having affinity to hTfR, and specificembodiments of combinations of the light chain and heavy chain of theantibody having affinity to hTfR, preferred examples of the amino acidsequence of the framework region 3 of the antibody heavy chain includethose having the amino acid sequence set forth as SEQ ID NO: 64.

Examples of preferred embodiments of humanized antibodies havingaffinity to hTfR include those having the amino acid sequences below:

anti-hTfR antibodies whose light chain variable region comprises theamino acid sequence set forth as SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, and whose heavychain variable region comprises the amino acid sequence set forth as SEQID NO: 65.

In the amino acid sequences of the light chain variable region set forthas SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, and SEQ ID NO: 22, CDR1 comprises the amino acid sequence setforth as SEQ ID NO: 6 or 7, CDR2 comprises the amino acid sequence setforth as SEQ ID NO: 8 or 9, and CDR3 comprises the amino acid sequenceset forth as SEQ ID NO: 10. However, the term CDRs as used above inregard to the amino acid sequences of the light chain variable regionset forth as SEQ ID NOs: 17 to 22 is not limited to those specificsequences but may also include a region containing the amino acidsequences of one of the CDRs or include an amino acid sequencecomprising not less than 3 consecutive amino acids of one of the aboveCDRs.

In the amino acid sequence of the heavy chain variable region set forthas SEQ ID NO: 65,

(a) CDR1 comprises the amino acid sequence set forth as SEQ ID NO: 62 or63,

(b) CDR2 comprises the amino acid sequence set forth as SEQ ID NO: 13 or14, and

(c) CDR3 comprises the amino acid sequence set forth as SEQ ID NO: 15 or16, and

the amino acid sequence set forth as SEQ ID NO: 64 is also contained asthe framework region 3. However, the term CDRs used above in regard tothe amino acid sequences of the heavy chain variable region set forth asSEQ ID NO:65 is not limited to those specific sequences but may alsoinclude a region containing the amino acid sequences of one of the CDRsor include an amino acid sequence comprising not less than 3 consecutiveamino acids of one of the above CDRs. The same also applies for theframework regions.

Examples of more specific embodiments of the humanized antibody havingaffinity to hTfR include:

the one that comprises the amino acid sequence set forth as SEQ ID NO:18 in the light chain variable region and comprises the amino acidsequence set forth as SEQ ID NO: 65 in the heavy chain variable region,

the one that comprises the amino acid sequence set forth as SEQ ID NO:20 in the light chain variable region and comprises the amino acidsequence set forth as SEQ ID NO: 65 in the heavy chain variable region,

the one that comprises the amino acid sequence set forth as SEQ ID NO:21 in the light chain variable region and comprises the amino acidsequence set forth as SEQ ID NO: 65 in the heavy chain variable region,and

the one that comprises the amino acid sequence set forth as SEQ ID NO:22 in the light chain variable region and comprises the amino acidsequence set forth as SEQ ID NO: 65 in the heavy chain variable region.

Examples of more specific embodiments of the humanized antibody havingaffinity to hTfR include:

the one that comprises the amino acid sequence set forth as SEQ ID NO:23 in the light chain and the amino acid sequence set forth as SEQ IDNO: 66 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:25 in the light chain and the amino acid sequence set forth as SEQ IDNO: 66 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:27 in the light chain and the amino acid sequence set forth as SEQ IDNO: 66 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:29 in the light chain and the amino acid sequence set forth as SEQ IDNO: 66 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:23 in the light chain and the amino acid sequence set forth as SEQ IDNO: 68 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:25 in the light chain and the amino acid sequence set forth as SEQ IDNO: 68 in the heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:27 in the light chain and the amino acid sequence set forth as SEQ IDNO: 68 in the heavy chain, and

the one that comprises the amino acid sequence set forth as SEQ ID NO:29 in the light chain and the amino acid sequence set forth as SEQ IDNO: 68 in the heavy chain.

In the above mentioned specific embodiments, the humanized anti-hTfRantibody comprising the amino acid sequence set forth as SEQ ID NO: 66in the heavy chain is an IgG1-type antibody, and the one comprising theamino acid sequence set forth as SEQ ID NO: 68 is an IgG4-type antibody.Both of them comprise the amino acid sequence set forth as SEQ ID NO: 65as the variable region.

Examples of more specific embodiments of the humanized antibody which isFab having affinity to hTfR include:

the one that comprises the amino acid sequence set forth as SEQ ID NO:23 in the light chain and comprises the amino acid sequence set forth asSEQ ID NO: 61 in the Fab heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:25 in the light chain and comprises the amino acid sequence set forth asSEQ ID NO: 61 in the Fab heavy chain,

the one that comprises the amino acid sequence set forth as SEQ ID NO:27 in the light chain and comprises the amino acid sequence set forth asSEQ ID NO: 61 in the Fab heavy chain, and

the one that comprises the amino acid sequence set forth as SEQ ID NO:29 in the light chain and comprises the amino acid sequence set forth asSEQ ID NO: 61 in the Fab heavy chain.

In the case where the anti-hTfR antibody is Fab, specific examples ofthe one in which a different Fc region is added to the Fab heavy chaininclude:

the one comprises the amino acid sequence set forth as SEQ ID NO: 23 inthe light chain and comprises the amino acid sequence set forth as SEQID NO: 71 in the Fab heavy chain with the Fc region added thereto,

the one comprises the amino acid sequence set forth as SEQ ID NO: 25 inthe light chain and comprises the amino acid sequence set forth as SEQID NO: 71 in the Fab heavy chain with the Fc region added thereto,

the one comprises the amino acid sequence set forth as SEQ ID NO: 27 inthe light chain and comprises the amino acid sequence set forth as SEQID NO: 71 in the Fab heavy chain with the Fc region added thereto, and

the one comprises the amino acid sequence set forth as SEQ ID NO: 29 inthe light chain and comprises the amino acid sequence set forth as SEQID NO: 71 in the Fab heavy chain with the Fc region added thereto.

When adding the above mentioned different Fc region to the Fab heavychain, for example, the Fab heavy chain with the Fc region added theretocan be linked, directly or via a linker sequence, to the differentprotein (A) on the C-terminal side thereof Besides, the one having theamino acid sequence set forth as SEQ ID NO: 71 is the one in which thehuman IgG Fc region having the amino acid sequence set forth as SEQ IDNO: 70 is linked, via a linker sequence comprising 25 amino acids thatis composed of consecutively linked five copies of the amino acidsequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), to the N-terminal side ofthe amino acid sequence of the Fab heavy chain (amino acid sequence: 61)of the humanized anti-hTfR antibody 3N.

Preferred embodiments of the antibody having affinity to hTfR have beenexemplified above. The light chain and heavy chain of those anti-hTfRantibodies may be mutated as desired, by substitution, deletion,addition and the like, in their variable-region amino acid sequences inorder to adjust the affinity of the anti-hTfR antibody to hTfR to asuitable level.

When replacing on or more amino acids of the light chain variable-regionamino acid sequence with other amino acids, the number of amino acids tobe replaced is preferably 1-10, more preferably 1-5, still morepreferably 1-3, and even more preferably 1-2. When deleting one or moreamino acids of the light chain variable-region amino acid sequence, thenumber of amino acids to be deleted is preferably 1-10, more preferably1-5, still more preferably 1-3, and even more preferably 1-2.Introduction of a combined mutation of such substitution and deletion ofamino acids is also allowed.

When adding one or more amino acids to the light chain variable region,they may be added inside, or on the N-terminal side or the C-terminalside of, the light chain variable-region amino acid sequence, andpreferably 1-10, more preferably 1-5, still more preferably 1-3, andeven more preferably 1-2, in number. Introduction of a combined mutationof such addition, substitution, and deletion of amino acids is alsoallowed. Such a mutated light chain variable-region amino acid sequencehas a homology preferably not lower than 80%, more preferably not lowerthan 90%, still more preferably not lower than 95%, to the amino acidsequence of the original light chain variable-region.

In particular, when replacing one or more amino acids of the amino acidsequence of respective CDRs or respective framework regions in the lightchain with other amino acids, the number of amino acids to be replacedis preferably 1-5, more preferably 1-3, still more preferably 1-2, andeven more preferably 1. When deleting one or more amino acid of theamino acid sequence of the respective CDRs or respective frameworkregions, the number of amino acids to be deleted is preferably 1-5, morepreferably 1-3, still more preferably 1-2, and even more preferably 1.Introduction of a combined mutation of such substitution and deletion ofamino acids is also allowed.

When adding one or more amino acids to the amino acid sequence ofrespective CDRs or respective framework regions in the light chain, theyare added inside, or on the N-terminal side or the C-terminal side of,the amino acid sequence, and preferably 1-5, more preferably 1-3, stillmore preferably 1-2, and even more preferably 1, in number. Introductionof a combined mutation of such addition, substitution, and deletion ofamino acids is also allowed. The amino acid sequence of each of suchmutated CDRs or framework regions has a homology preferably not lowerthan 80%, more preferably not lower than 90%, and still more preferablynot lower than 95%, to the amino acid sequence of the respectiveoriginal CDRs.

When replacing one or more amino acids of the amino acid sequence setforth as SEQ ID NO: 65 which is the heavy chain variable region withother amino acids, the number of amino acids to be replaced ispreferably 1-10, more preferably 1-5, still more preferably 1-3, andeven more preferably 1-2. When deleting one or more amino acids of theheavy chain variable-region amino acid sequence, the number of aminoacids to be deleted is preferably 1-10, more preferably 1-5, still morepreferably 1-3, and even more preferably 1-2. Introduction of a combinedmutation of such substitution and deletion of amino acids is alsoallowed.

When adding one or more amino acids to the amino acid sequence set forthas SEQ ID NO: 65 which is the heavy chain variable region, they may beadded inside, or on the N-terminal side or the C-terminal side of, theheavy chain variable-region amino acid sequence, and preferably 1-10,more preferably 1-5, still more preferably 1-3, and even more preferably1-2, in number. Introduction of a combined mutation of such addition,substitution, and deletion of amino acids is also allowed. Such amutated heavy chain variable-region amino acid sequence has a homologypreferably not lower than 80%, more preferably not lower than 90%, stillmore preferably not lower than 95%, to the amino acid sequence of theoriginal heavy chain variable-region.

In particular, when replacing one or more amino acids of the amino acidsequence of respective CDRs or respective framework regions in the aminoacid sequence set forth as SEQ ID NO: 65 with other amino acids, thenumber of amino acids to be replaced is preferably 1-5, more preferably1-3, still more preferably 1-2, and even more preferably 1. Whendeleting one or more amino acid of the amino acid sequence of therespective CDRs, the number of amino acids to be deleted is preferably1-5, more preferably 1-3, still more preferably 1-2, and even morepreferably 1. Introduction of a combined mutation of such substitutionand deletion of amino acids is also allowed.

When adding one or more amino acids to the amino acid sequence ofrespective CDRs or respective framework regions in the amino acidsequence set forth as SEQ ID NO: 65, they are added inside, or on theN-terminal side or the C-terminal side of, the amino acid sequence, andpreferably 1-5, more preferably 1-3, still more preferably 1-2, and evenmore preferably 1 in number. Introduction of a combined mutation of suchaddition, substitution, and deletion of amino acids is also allowed. Theamino acid sequence of each of such mutated CDRs has a homologypreferably not lower than 80%, more preferably not lower than 90%, andstill more preferably not lower than 95%, to the amino acid sequence ofthe respective original CDRs.

Besides, in the case where the mutation described above such assubstitution, deletion, addition is introduced to the amino acidsequence set forth as SEQ ID NO: 65 which is the heavy chain variableregion of the anti-hTfR antibody, it is preferable that the amino acidmethionine at position 5 from the N-terminal side of the original CDR1set forth as SEQ ID NO: 62 or 63, and the amino acid leucine at position17 from the N-terminal side of the framework region 3 set forth as SEQID NO: 64 should be conserved at the same position as the original ones.Further, it is preferable that the amino acid sequences of the heavychain CDR1 and framework region 3 should also be conserved at the samepositions as the original ones.

A mutation may be introduced into both the variable regions of the lightchain and the heavy chain of the anti-hTfR antibody, by combining theabove mutation into the light chain variable region of the anti-hTfRantibody and the above mutation into the heavy chain variable region ofthe anti-hTfR antibody.

Examples of the above mentioned substitution of one or more amino acidsin the amino acid sequence of the variable regions of the heavy chainand the light chain of the anti-hTfR antibody include amino acidsclassified into the same groups, such as aromatic amino acids (Phe, Trp,Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids(Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu,Asp), amino acids having a hydroxy group (Ser, Thr).

Besides, in the case where introducing a mutation into the anti-hTfRantibody by adding one or more amino acids on its C-terminus or theN-terminus, if the added amino acids are positioned between theanti-hTfR antibody and a different protein (A) when they are fused, theadded amino acids constitute part of a linker.

In the above preferred embodiments of the antibody, including humanizedantibody, having affinity to hTfR, there is no particular limitation asto the amino acid sequence of the anti-hTfR antibody heavy chain andlight chain CDRs, insofar as the antibody has a specific affinity tohTfR and monkey TfR.

However, in the present invention, a human antibody having a relativelyhigh affinity to hTfR and has affinity both to human and monkey TfRs,simultaneously, is one

whose dissociation constant with human TfR is preferably not greaterthan 1×10⁻¹⁰ M, more preferably not greater than 2.5×10⁻¹¹ M, still morepreferably not greater than 5×10⁻¹² M, and even more preferably notgreater than 1×10⁻¹² M, and

whose dissociation constant with monkey TfR is preferably not greaterthan 1×10⁻⁹ M, more preferably not greater than 5×10⁻¹⁰ M, and stillmore preferably not greater than 1×10⁻¹⁰ M, for example, not greaterthan 7.5×10⁻¹¹ M,

in particular, as measured by the method described in Example 7.

For example, the dissociation constants with human TfR and monkey TfRare not greater than 1×10⁻¹⁰ M and not greater than 1×10⁻⁹ M, notgreater than 1×10⁻¹¹ M and not greater than 5×10⁻¹⁰ M, not greater than5×10⁻¹² M and not greater than 1×10⁻¹⁰ M, not greater than 5×10⁻¹² M andnot greater than 7.5×10⁻¹¹ M, not greater than 1×10⁻¹² M and not greaterthan 1×10⁻¹⁰ M, or not greater than 1×10⁻¹² M and not greater than7.5×10⁻¹¹ M, respectively. In this context, although there is noparticularly definite lower limit on the dissociation constant withhuman TfR, it can be, for example, 5×10⁻¹³ M or 1×10⁻¹³ M. Further,although there is no particularly definite lower limit on thedissociation constant with monkey TfR, it can be, for example, 5×10⁻¹¹M, 1×10⁻¹¹ M, 1×10⁻¹² M, and so on. The same applies when the antibodyis a single-chain antibody.

Specific embodiments of the fusion protein between the humanizedantibody, which has affinity to hTfR, and a different protein (A)described above include: those in which the different protein (A) ishuman acidic α-glucosidase (hGAA), human iduronate 2-sulfatase (hI2S),human α-L-iduronidase (hIDUA), human palmitoyl protein thioesterase 1(hPPT-1), human acidic sphingomyelinase (hASM), human arylsulfatase A(hARSA), human heparan N-sulfatase (hSGSH), human glucocerebrosidase(hGBA), human tripeptidyl-peptidase 1 (hTPP-1), humanα-N-acetylglucosaminidase (hNAGLU), human β-glucuronidase (hGUSB), humanacid ceramidase (hAC), human α-L-fucosidase (hFUCA1), or α-mannosidase(hLAMAN).

Specific examples of the fusion protein where the different protein (A)is human acidic α-glucosidase (hGAA) include:

(1) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 27,

(4) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,hGAA is one having the amino acid sequence set forth as SEQ ID NO: 55 or56, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is human acidic α-glucosidase (hGAA) include:

(1) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side thereof and via the amino acidsequence Gly-Ser, to hGAA, and the other portion consisting of the hTfRlight chain, wherein the amino acid sequence of the former is set forthas SEQ ID NO: 57, and the amino acid sequence of the latter is set forthas SEQ ID NO: 23, and

(2) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side thereof and via the amino acidsequence Gly-Ser, to hGAA, and the other portion consisting of the hTfRlight chain, wherein the amino acid sequence of the former is set forthas SEQ ID NO: 58, and the amino acid sequence of the latter is set forthas SEQ ID NO: 23.

The antibody of (1) is of IgG1 type, and the antibody of (2) is of IgG4type. Further, hGAA is one having the amino acid sequence set forth asSEQ ID NO: 55.

In (1) above, the amino acid sequence of the hTfR heavy chain, which isincluded in SEQ ID NO: 57, is the one set forth as SEQ ID NO: 66.Namely, the fusion protein according to (1) above, includes, as ahumanized antibody, the amino acid sequence of the light chain set forthas SEQ ID NO: 23 and the amino acid sequence of the heavy chain setforth as SEQ ID NO: 66. In (2) above, the amino acid sequence of thehTfR heavy chain, which is included in SEQ ID NO: 58, is the one setforth as SEQ ID NO: 68. Namely, the fusion protein according to (2)above, includes, as a humanized antibody, the amino acid sequence of thelight chain set forth as SEQ ID NO: 23 and the amino acid sequence ofthe heavy chain set forth as SEQ ID NO: 68.

Specific examples of the fusion protein where the different protein (A)is human acidic α-glucosidase (hGAA) and the humanized antibody is a Fabantibody include:

(1) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 27,

(4) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hGAA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

A more specific example of the fusion protein where the differentprotein (A) is human acidic α-glucosidase (hGAA) and the humanizedantibody is a Fab antibody includes: the one that is composed of theportion consisting of hTfR Fab heavy chain linked, on the C-terminalside thereof and via a linker sequence, that linker sequence consists ofthree consectively linked amino acid sequences each set forth as SEQ IDNO: 3, to hGAA, and the other portion consisting of the hTfR lightchain, wherein the amino acid sequence of the former is set forth as SEQID NO: 89, and the amino acid sequence of the latter is set forth as SEQID NO: 23.

The fusion protein where the different protein (A) is human acidicα-glucosidase (hGAA) has affinity both to human and monkey TfRs,simultaneously, and is one

whose dissociation constant with monkey TfR is preferably not greaterthan 1×10⁻¹⁰ M, more preferably not greater than 5×10⁻¹¹ M,

whose dissociation constant with human TfR is preferably not greaterthan 1×10⁻¹⁰ M, more preferably not greater than 5×10⁻¹¹ M, still morepreferably not greater than 1×10⁻¹¹ M, and even more preferably notgreater than 1×10⁻¹² M,

as measured by the method described in Example 7.

For example, the dissociation constants with monkey TfR and human TfRare not greater than 1×10⁻¹⁰ M and not greater than 1×10⁻¹⁰ M, notgreater than 1×10⁻¹⁰ M and not greater than 1×10⁻¹¹ M, not greater than1×10⁻¹⁰ M and not greater than 1×10⁻¹² M, not greater than 5×10⁻¹¹ M andnot greater than 1×10⁻¹¹ M, not greater than 5×10⁻¹¹ M and not greaterthan 1×10⁻¹¹ M, or not greater than 5×10⁻¹¹ M and not greater than1×10⁻¹² M, respectively. In this context, although there is noparticularly definite lower limit on the dissociation constant withmonkey TfR, it can be, for example, 1×10⁻¹¹ M, 1×10⁻¹² M, or 1×10⁻¹³ M.Although there is no particularly definite lower limit on thedissociation constant with human TfR, it can be, for example, 1×10⁻¹² M,5×10⁻¹³ M, or 1×10⁻¹³ M. The same also applies if the antibody is asingle-chain antibody.

Specific examples of the fusion protein where the different protein (A)is human iduronate 2-sulfatase (hI2S) include:

(1) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 27,

(4) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the heavy chain isset forth as SEQ ID NO: 66 or 68, and the amino acid sequence of thelight chain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type.

More specific examples of the fusion protein where the different protein(A) is human iduronate 2-sulfatase (hI2S) include:

the one that is composed of: the portion consisting of hTfR heavy chainlinked, on the C-terminal side thereof and via the amino acid sequenceGly-Ser, to hI2S, and the other portion consisting of the hTfR lightchain, wherein the amino acid sequence of the former is set forth as SEQID NO: 53, and the amino acid sequence of the latter is set forth as SEQID NO: 23. In this case, the antibody is of IgG1 type. The amino acidsequence of the hTfR heavy chain, which is included in SEQ ID NO: 53, isthe one set forth as SEQ ID NO: 66. Namely, this fusion proteinincludes, as a humanized antibody, the amino acid sequence of the lightchain set forth as SEQ ID NO: 23 and the amino acid sequence of theheavy chain set forth as SEQ ID NO: 66. The antibody may be of IgG4type, and in this case, the amino acid sequence of the heavy chain setforth as SEQ ID NO: 66 may be replaced with the amino acid sequence setforth as SEQ ID NO: 68.

Specific examples of the fusion protein where the different protein (A)is human iduronate 2-sulfatase (hI2S) and the humanized antibody is aFab antibody include:

(1) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to hI2S, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the Fab heavy chainis set forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 27,

(4) the one that is composed of the portion consisting of hTfR Fab heavychain linked, on the C-terminal side or the N-terminus thereof and via alinker sequence, to hI2S, and the other portion consisting of the hTfRlight chain, wherein the amino acid sequence of the Fab heavy chain isset forth as SEQ ID NO: 61, and the amino acid sequence of the lightchain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

Specific examples of the fusion protein where the different protein (A)is hIDUA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hIDUA is one having the amino acid sequence set forth as SEQ ID NO:75 or 76, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hIDUA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hIDUA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 90, and the amino acid sequence of the latter is setforth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkercomprising 42 amino acids in total that consists of the amino acidsequence Gly-Ser followed by consecutively linked eight copies of theamino acid sequence set forth as SEQ ID NO: 3, to hIDUA, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the former is set forth as SEQ ID NO: 91, and the amino acidsequence of the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hIDUA, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 92, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 93, and the amino acid sequence ofthe latter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 94, and the amino acid sequence ofthe latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 95, and the amino acid sequence ofthe latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hIDUA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 96, and the amino acid sequence ofthe latter is set forth as SEQ ID NO: 23,

(8) the one that is composed of: the portion consisting of hIDUA linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 97, and theamino acid sequence of the latter is set forth as SEQ ID NO: 23, and

(9) the one that is composed of: the portion consisting of hIDUA linkedto single-chain humanized anti-hTfR antibody No. 3N(2) set forth as SEQID NO: 98, on the C-terminal side thereof and via a linker comprisingseven amino acids that consists of the amino acid sequence Gly-Glyfollowing the amino acid sequence set forth as SEQ ID NO: 3, and that isset forth as SEQ ID NO: 99.

The fusion proteins (1) to (3) are those whose antibody is of IgG1 type,and the fusion proteins (4) to (9) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (3) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (3) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (4) to (9) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (4) to (9) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hPPT-1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hPPT-1 is one having the amino acid sequence set forth as SEQ ID NO:77, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hPPT-1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hPPT-1, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 100, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 101, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hPPT-1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 102, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hPPT-1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 103, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hPPT-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 104, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(6) the one that is composed of the portion consisting of hPPT-1 linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 105, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) and (6) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) and (6) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) and (6) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hASM include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hASM is one having the amino acid sequence set forth as SEQ ID NO:78, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hASM include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hASM, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 106, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 107, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hASM, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 108, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hASM, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 109, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 110, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 111, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 112, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hASM, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 113, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(9) the one that is composed of: the portion consisting of hASM linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 114, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (9) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) and (6) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) and (6) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hARSA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hARSA is one having the amino acid sequence set forth as SEQ ID NO:79, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hARSA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hARSA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 115, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 116, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hARSA, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 117, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hARSA, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 118, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 119, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 120, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 121, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hARSA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 122, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(9) the one that is composed of: the portion consisting of hARSA linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 123, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (9) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (9) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (9) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hSGSH include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hSGSH is one having the amino acid sequence set forth as SEQ ID NO:80, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hSGSH include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hSGSH, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 124, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 125, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hSGSH, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 126, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hSGSH, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 127, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hSGSH, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 128, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(6) the one that is composed of the portion consisting of hSGSH linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 129, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) and (6) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) and (6) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) and (6) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hGBA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hGBA is one having the amino acid sequence set forth as SEQ ID NO:81, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hGBA include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hGBA, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 130, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 131, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGBA, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 132, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGBA, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 133, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGBA, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 134, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(6) the one that is composed of: the portion consisting of hGBA linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 135, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) and (6) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) and (6) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) and (6) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hTPP-1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hTPP-1 is one having the amino acid sequence set forth as SEQ ID NO:82, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hTPP-1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hTPP-1, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 136, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 137, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hTPP-1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 138, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hTPP-1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 139, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hTPP-1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 140, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(6) the one that is composed of the portion consisting of hTPP-1 linkedto the amino acid sequence on the C-terminal side thereof that consistsof consecutively linked two copies of the hTfR heavy chain (Fab) having,on the C-terminal side thereof, the linker consisting of consecutivelylinked six copies of the amino acid sequence set forth as SEQ ID NO: 3,and the other portion consisting of the hTfR light chain, wherein theamino acid sequence of the former is set forth as SEQ ID NO: 141, andthe amino acid sequence of the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) and (6) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) and (6) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) and (6) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hNAGLU include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hNAGLU is one having the amino acid sequence set forth as SEQ ID NO:83, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hNAGLU include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hNAGLU, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 142, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 143, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hNAGLU, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 144, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hNAGLU, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 145, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 146, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 147, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 148, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hNAGLU, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 149, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hGUSB include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hGUSB is one having the amino acid sequence set forth as SEQ ID NO:84, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hGUSB include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hGUSB, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 150, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 151, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGUSB, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 152, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGUSB, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 153, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 154, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 155, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 156, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGUSB, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 157, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hGALC include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hGALC is one having the amino acid sequence set forth as SEQ ID NO:85, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hGALC include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hGALC, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 158, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 159, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGALC, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 160, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hGALC, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 161, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 162, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 163, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 164, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hGALC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 165, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hAC include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hAC is one having the amino acid sequence set forth as SEQ ID NO:86, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hAC include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hAC, and the other portion consisting of thehTfR light chain, wherein the amino acid sequence of the former is setforth as SEQ ID NO: 166, and the amino acid sequence of the latter isset forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 167, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hAC, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 168, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hAC, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 169, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 170, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 171, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 172, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hAC, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 173, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hFUCA1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hFUCA1 is one having the amino acid sequence set forth as SEQ ID NO:87, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hFUCA1 include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hFUCA1, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 174, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 175, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hFUCA1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 176, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hFUCA1, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 177, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 178, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 179, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 180, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hFUCA1, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 181, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is hLAMAN include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 27, and

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side or the N-terminal sidethereof and via a linker sequence, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe heavy chain is set forth as SEQ ID NO: 66 or 68, and the amino acidsequence of the light chain is set forth as SEQ ID NO: 29. Here, it ispreferable that the linker sequence is one consisting of a singleglycine, a single serine, the amino acid sequence Gly-Ser, the aminoacid sequence Gly-Gly-Ser, the amino acid sequence set forth as SEQ IDNO: 3, the amino acid sequence set forth as SEQ ID NO: 4, the amino acidsequence set forth as SEQ ID NO: 5, and the amino acid sequencesconsisting of 1 to 10 or 1 to 20 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type. Further,the hLAMAN is one having the amino acid sequence set forth as SEQ ID NO:88, or a mutant thereof.

More specific examples of the fusion protein where the different protein(A) is hLAMAN include:

(1) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via the aminoacid sequence Gly-Ser, to hLAMAN, and the other portion consisting ofthe hTfR light chain, wherein the amino acid sequence of the former isset forth as SEQ ID NO: 182, and the amino acid sequence of the latteris set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 183, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(3) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked ten copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hLAMAN, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 184, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(4) the one that is composed of: the portion consisting of the hTfRheavy chain linked, on the C-terminal side thereof and via a linkerconsisting of consecutively linked 20 copies of the amino acid sequenceset forth as SEQ ID NO: 3, to hLAMAN, and the other portion consistingof the hTfR light chain, wherein the amino acid sequence of the formeris set forth as SEQ ID NO: 185, and the amino acid sequence of thelatter is set forth as SEQ ID NO: 23,

(5) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked three copies of the amino acidsequence set forth as SEQ ID NO: 3, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 186, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(6) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked five copies of the amino acidsequence set forth as SEQ ID NO: 3, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 187, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23,

(7) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked ten copies of the amino acidsequence set forth as SEQ ID NO: 3, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 188, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23, and

(8) the one that is composed of: the portion consisting of the hTfRheavy chain (Fab) linked, on the C-terminal side thereof and via alinker consisting of consecutively linked 20 copies of the amino acidsequence set forth as SEQ ID NO: 3, to hLAMAN, and the other portionconsisting of the hTfR light chain, wherein the amino acid sequence ofthe former is set forth as SEQ ID NO: 189, and the amino acid sequenceof the latter is set forth as SEQ ID NO: 23.

The fusion proteins (1) to (4) are those whose antibody is of IgG1 type,and the fusion proteins (5) to (8) are those whose antibody is of Fabtype.

Here, the amino acid sequences of the hTfR heavy chains in the abovefusion proteins (1) to (4) are set forth as SEQ ID NO: 68. Namely, theabove fusion proteins (1) to (4) have a humanized antibody whose lightchain includes the amino acid sequence set forth as SEQ ID NO: 23 andwhose heavy chain includes the amino acid sequence set forth as SEQ IDNO: 68. Further, the amino acid sequences of the hTfR heavy chains inthe above fusion proteins (5) to (8) are set forth as SEQ ID NO: 61.Namely, the above fusion proteins (5) to (8) have a humanized antibodywhose light chain includes the amino acid sequence set forth as SEQ IDNO: 23 and whose heavy chain (Fab) includes the amino acid sequence setforth as SEQ ID NO: 61.

Specific examples of the fusion protein where the different protein (A)is a human lysosomal enzyme include:

(1) the one that is composed of the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the heavy chain is set forth as SEQ ID NO: 66 or 68, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the heavy chain is set forth as SEQ ID NO: 66 or 68, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the heavy chain is set forth as SEQ ID NO: 66 or 68, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 27,

(4) the one that is composed of: the portion consisting of hTfR heavychain linked, on the C-terminal side or the N-terminal side thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the heavy chain is set forth as SEQ ID NO: 66 or 68, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

Here, the antibody having SEQ ID NO: 66 in the heavy chain is of IgG1type, and the antibody having SEQ ID NO: 68 is of IgG4 type.

Specific examples of the fusion protein where the different protein (A)is a human lysosomal enzyme and the humanized antibody is a Fab antibodyinclude:

(1) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the Fab heavy chain is set forth as SEQ ID NO: 61, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 23,

(2) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the Fab heavy chain is set forth as SEQ ID NO: 61, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 25,

(3) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the Fab heavy chain is set forth as SEQ ID NO: 61, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 27,

(4) the one that is composed of: the portion consisting of hTfR Fabheavy chain linked, on the C-terminal side or the N-terminus thereof andvia a linker sequence, to the human lysosomal enzyme, and the otherportion consisting of the hTfR light chain, wherein the amino acidsequence of the Fab heavy chain is set forth as SEQ ID NO: 61, and theamino acid sequence of the light chain is set forth as SEQ ID NO: 29.

Here, it is preferable that the linker sequence is one consisting of asingle glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence set forth asSEQ ID NO: 3, the amino acid sequence set forth as SEQ ID NO: 4, theamino acid sequence set forth as SEQ ID NO: 5, and the amino acidsequences consisting of 1 to 10 thereof that are consecutively linked.

A human lysosomal enzyme other than above can also be prepared into afusion protein with the hTfR antibody according to the same embodimentsas those described about hGAA and hI2S. Examples of the human lysosomalenzyme include β-galactosidase, GM2 activator protein, β-hexosaminidaseA, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase,β-mannosidase, galactosylceramidase, saposin C, aspartylglucosaminidase,α-galactosidase A, α-N-acetylglucosaminidase, acetyl CoA:α-glucosaminideN-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase,amylo-1,6-glucosidase, sialidase, hyaluronidase 1, CLN1, and CLN2,though not limited thereto. Also, when fusing a protein other than thelysosome, the fusion protein may be constructed in the same manner.

The fusion protein where the different protein (A) is a human lysosomalenzyme has an affinity to both human TfR and monkey TfR, and has thefollowing dissociation constant as measured by the method described inExample 7:

dissociation constant with monkey TfR: preferably not greater than1×10⁻¹⁰ M, more preferably not greater than 5×10⁻¹¹ M; and

dissociation constant with human TfR: preferably not greater than1×10⁻¹⁰ M, more preferably not greater than 5×10⁻¹¹ M, still morepreferably not greater than 1×10⁻¹¹ M, even more preferably not greaterthan 1×10⁻¹² M.

For example, the dissociation constants with monkey TfR and human TfRare not greater than 1×10⁻¹⁰ M and not greater than 1×10⁻¹⁰ M, notgreater than 1×10⁻¹⁰ M and not greater than 1×10⁻¹¹ M, not greater than1×10⁻¹⁰ M and not greater than 1×10⁻¹² M, not greater than 5×10⁻¹¹ M andnot greater than 1×10⁻¹¹ M, not greater than 5×10⁻¹¹ M and not greaterthan 1×10⁻¹¹ M, or not greater than 5×10⁻¹¹ M and not greater than1×10⁻¹² M, respectively. In this context, although there is noparticularly definite lower limit on the dissociation constant withmonkey TfR, it can be, for example, 1×10⁻¹¹ M, 1×10⁻¹² M or 1×10⁻¹³ M.Although there is no particularly definite lower limit on thedissociation constant with human TfR, it can be, for example, 1×10⁻¹² M,5×10⁻¹³ M or 1×10⁻¹³ M. The same also applies if the antibody is asingle-chain antibody.

The anti-hTfR antibody according to the present invention can be usedfor the production of a pharmaceutical agent for parenteraladministration for the treatment of a disease condition of the centralnervous system by binding it to the molecule of a physiologically activeprotein or a pharmacologically active low-molecular-weight compound. Andthe anti-hTfR antibody conjugated with the molecule of a physiologicallyactive protein or a pharmacologically active low-molecular-weightcompound, can be used in the method of treatment of a patient with adisease condition of the central nervous system, in which atherapeutically effective amount of a physiologically active protein orpharmacologically active low-molecular-weight compound is administeredto the patient with a disease of the central nervous system parenterally(including intravenous injection such as intravenous infusion). Theanti-hTfR antibody conjugated with the molecule of a physiologicallyactive protein or a pharmacologically active low-molecular-weightcompound, after parenterally administered, can not only get inside thebrain but also reach other organs where hTfR is expressed. Further, thepharmaceutical agent may be used for preventing the onset of a disease.

In particular, as the anti-hTfR antibody of the present invention can,as a conjugate with human acidic α-glucosidase (hGAA), enable hGAA topass through the blood-brain barrier and function in the brain, theantibody can be used for the production of a pharmaceutical agent forparenteral administration for the treatment of a disease condition ofthe central nervous system accompanying Pompe's disease. Further, theanti-hTfR antibody conjugated with hGAA can be used in the method oftreatment of a patient with a disease condition of the central nervoussystem disorder accompanying Pompe's disease, in which a therapeuticallyeffective amount of the antibody is administered to the patient withPompe's disease parenterally (including intravenous injection such asintravenous infusion). The anti-hTfR antibody conjugated with hGAA,after parenterally administered, can not only get inside the brain butalso reach other organs where hTfR is expressed. Further, thepharmaceutical agent may be used for preventing the onset of the diseasecondition.

In particular, as the anti-hTfR antibody of the present invention can,as a conjugate with human iduronate 2-sulfatase (hI2S), enable hI2S topass through the blood-brain barrier and function in the brain, theantibody can be used for the production of a pharmaceutical agent forparenteral administration for the treatment of a disease condition ofthe central nervous system accompanying Hunter syndrome. Further, theanti-hTfR antibody conjugated with hI2S can be used in the method oftreatment of a patient with a disease condition of the central nervoussystem disorder accompanying Hunter syndrome, in which a therapeuticallyeffective amount of the antibody is administered to the patient withHunter syndrome parenterally (including intravenous injection such asintravenous infusion). The anti-hTfR antibody conjugated with hI2S,after parenterally administered, can not only get inside the brain butalso reach other organs where hTfR is expressed. Further, thepharmaceutical agent may be used for preventing the onset of thedisorders.

In particular, as the anti-hTfR antibody of the present invention can,as a conjugate with human α-L-iduronidase (hIDUA), enable hIDUA to passthrough the blood-brain barrier and function in the brain, the antibodycan be used for production of a pharmaceutical agent for parenteraladministration for the treatment of a disease condition of the centralnervous system accompanying the Hurler syndrome. Further, the anti-hTfRantibody conjugated with hIDUA can be used in the method of treatment ofa patient with a disease condition of the central nervous systemdisorder accompanying the Hurler syndrome, in which a therapeuticallyeffective amount of the antibody is administered to the patient withHurler syndrome parenterally (including intravenous injection such asintravenous infusion). The anti-hTfR antibody conjugated with hIDUA,after parenterally administered, can not only get inside the brain butalso reach other organs where hTfR is expressed. The pharmaceuticalagent can also be used for prophylaxis of such disease conditions.

Further, in particular, as the anti-hTfR antibody of the presentinvention can, as a conjugate with a human lysosomal enzyme, enable thehuman lysosomal enzyme to pass through the blood-brain barrier andfunction in the brain, the antibody can be used for the production of apharmaceutical agent for parenteral administration for the treatment ofa disease condition of central nervous system caused by deficiency inthe human lysosomal enzyme. Further, the anti-hTfR antibody conjugatedwith the human lysosomal enzyme can be used in the method of treatmentof a patient with a disease condition of the central nervous systemcaused by deficiency in the human lysosomal enzyme, in which atherapeutically effective amount of the antibody is administered to thepatient parenterally (including intravenous injection such asintravenous infusion). The anti-hTfR antibody conjugated with the humanlysosomal enzyme, after parenterally administered, can not only getinside the brain but also reach other organs where the human lysosomalenzyme is expressed. Further, the pharmaceutical agent may be used forpreventing the onset of the disease condition.

The proteins, low-molecular-weight compound and the like that areconjugated with the anti-hTfR antibody of the present invention can beused as pharmaceutical agents which are to exhibit their functions inthe central nervous system (CNS) after parenterally administered. Suchpharmaceutical agents may be administered to patients generally byintravenous injection such as intravenous injection, subcutaneousinjection, intramuscular injection and the like, though there is noparticular limitation as to the route of their administration.

The proteins, the low-molecular-weight compounds and the like that areconjugated with the anti-hTfR antibody of the present invention can beprovided to medical facilities as pharmaceutical agents in such forms oflyophilized product or aqueous preparation. In the case of an aqueouspreparation, it can be provided in the form of preparations in which oneof the pharmaceutical agents is dissolved in a solution containing astabilizer, buffer, and an isotonizer in advance, and sealed in vials orsyringes. A type of preparations sealed in a syringe is generally calleda prefilled syringe-type preparation. Taking the form of a prefilledsyringe-type preparation facilitates patients' self-administration ofthe pharmaceutical agent.

Where an aqueous preparation is provided, the concentration of theprotein, the low-molecular-weight compound or the like conjugated withthe anti-hTfR antibody in the aqueous preparation is, e.g., 1-4 mg/mL,though it is to be adjusted as desired in accordance with the dosage.Where there is no particular limitation as to stabilizers to becontained in the aqueous preparation insofar as they arepharmaceutically available, nonionic surfactants may preferably be used.Examples of such nonionic surfactants include polysorbate and poloxamer,either of which may be used alone or in combination. Among polysorbates,polysorbate 20 and polysorbate 80 are preferably used. As poloxamer,poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) isparticularly preferred. Further, the concentration of nonionicsurfactant contained in the aqueous preparation is preferably 0.01-1mg/mL, more preferably, 0.01-0.5 mg/mL, and still more preferably0.1-0.5 mg/mL. As stabilizers, amino acids such as histidine, arginine,methionine, and glycine may also be used. Where employed as astabilizer, the concentration of an amino acid in the aqueouspreparation is preferably 0.1-40 mg/mL, more preferably 0.2-5 mg/mL, andstill more preferably 0.5-4 mg/mL. While there is no particularlimitation as to a buffer to be contained in the aqueous preparationinsofar as it is pharmaceutically available, phosphate buffer ispreferred, and more preferred is sodium phosphate buffer. Where used asa buffer, the concentration of sodium phosphate is preferably 0.01-0.04M. The pH of the aqueous preparation adjusted with a buffer ispreferably 5.5-7.2. While there is no particular limitation as to anisotonizer to be contained in the aqueous preparation insofar as it ispharmaceutically available, sodium chloride or mannitol may bepreferably used alone or in combination as an isotonizer.

EXAMPLES

Though the present invention is described in further detail below withreference to examples, it is not intended that the present invention belimited to those examples. Examples 1 to 15 are about Reference Example(antibody No. 3).

Example 1 Construction of hTfR Expression Vector

Employing human spleen Quick Clone cDNA (Clontech Inc.) as a templateand using primer hTfR5′ (SEQ ID NO:41) and primer hTfR3′ (SEQ ID NO:42),PCR was performed to amplify the gene fragment encoding humantransferrin receptor (hTfR). The amplified fragment encoding hTfR wasdigested with MluI and NotI, and then inserted between MluI and NotIsites of vector pCI-neo (Promega Inc.). The vector thus prepared wasdesignated pCI-neo(hTfR). This vector then was digested with MluI andNotI to cut out the gene fragment encoding hTfR, and this fragment wasinserted between MluI and NotI sites of pE-mIRES-GS-puro, an expressionvector disclosed in an international publication WO 2012/063799 toconstruct an hTfR expression vector, pE-mIRES-GS-puro(hTfR).

Example 2 Preparation of Recombinant hTfR

Into CHO-K1 cells was introduced pE-mIRES-GS-puro(hTfR) byelectroporation, and the cells then were subjected to selection culturein a CD OptiCHO™ medium (Invitrogen Inc.) containing methioninesulfoximine (MSX) and puromycin to prepare recombinant hTfR expressingcells. The recombinant hTfR expressing cells were cultured, andrecombinant hTfR was prepared.

Example 3 Immunization of Mouse with Recombinant hTfR

Mice were immunized with recombinant hTfR prepared in Example 2 asantigen. Immunization was carried out by intravenously orintraperitoneally injecting the mice with the antigen.

Example 4 Preparation of Hybridoma Cells

About one week after the last injection, the spleens of the mice wereexcised and homogenized to isolate spleen cells. The spleen cells thusobtained were fused with cells of mouse myeloma cell line(P3.X63.Ag8.653) by the polyethylene glycol method. After cell fusion,the cells were suspended in a RPMI 1640 medium containing (1×) HATsupplement (Life Technologies Inc.) and 10% Ultra low IgG fetal bovineserum (Life Technologies Inc.), and the cell suspension was dispensed to20 of 96-well plates, 200 μL/well. After the cells were cultured for 10days in a carbon dioxide gas incubator (37° C., 5% CO₂), each well wasexamined under a microscope, and the wells that contain a single colonywere selected.

When the cells in each well reached near confluence, the culturesupernatant was collected as a culture supernatant of hybridoma, andsubjected to the following screening process.

Example 5 Screening of High Affinity Antibody Producing Cell Line

The recombinant hTfR solution (Sino Biologics Inc.) was diluted with 50mM sodium phosphate buffer (pH 9.5-9.6) to 5 μg/mL to prepare a solidphase solution. After 50 μL of the solid phase solution was added toeach well of a Nunc MaxiSorp™ flat-bottom 96-well plate (substrate:polystyrene, mfd. by Nunc Inc.), the plate was left to stand for onehour at room temperature to let the recombinant hTfR adhere to the plateand become immobilized. The solid phase solution was discarded, eachwell was washed three times with 250 μL of washing solution (PBScontaining PBS-T: 0.05% Tween20), 200 μL of a blocking solution (PBScontaining 1% BSA) then was added to each well, and the plate was leftto stand for one hour at room temperature.

The blocking solution was discarded, and each well was washed threetimes with 250 μL of PBS-T. To each well was added 50 μL of thehybridoma culture supernatant, and the plate was left to stand for onehour at room temperature to let the mouse anti-hTfR antibody containedin the culture supernatant bind to the recombinant hTfR. At the sametime, to some wells was added 50 μL of culture supernatant of ahybridoma that did not produce mouse anti-hTfR antibody, as a control.In addition, 50 μL of the medium for hybridoma culture was added to thewells, as mock wells, beside those wells to which the culturesupernatant was added. Measurement was conducted in an n=2 fashion.Then, the solution was discarded, and each well was washed three timeswith 250 μL of PBS-T.

To each of the above wells was added 100 μL of HRP-labelled goatanti-mouse immunoglobulin antibody solution (Promega Inc.), and theplate was left to stand for 30 minutes at room temperature. The solutionthen was discarded, and each well was washed three times with 250 μL ofPBS-T. To each well as added 50 μL of a chromogenic substrate solution,TMB Stabilized Substrate for Horseradish Peroxidase (Promega Inc.), andthe wells were left to stand for 10 to 20 minutes at room temperature.Then, following addition of 100 μL of a stop solution (2N sulfuricacid), the absorbance of each well was measured on a plate reader at 450nm. Of the two wells for each of the culture supernatant and control,the mean values were taken, respectively, and from each of the meanvalues, the respective mean value for the two mock wells placedcorresponding to each of the culture supernatant and the control, wassubtracted, giving the measurement.

Fourteen types of hybridoma cells corresponding to culture supernatantsadded to the wells which exhibited the higher measurements were selectedas the cell lines (high affinity antibody producing cell line) thatproduce antibodies exhibiting high affinities to hTfR (high affinityanti-hTfR antibody). These fourteen types of cell lines were designatedas Clone 1 line to Clone 14 line. Clone 3 line was selected from thesecell lines and used in the experiments below. Further, the anti-hTfRantibody produced by Clone 3 line was designated as anti-hTfR antibodyNo. 3.

Example 6 Analysis of the Variable-Region Amino Acid Sequence of theHigh Affinity Anti-hTfR Antibodies

From the Clone 3 line selected in Example 5, cDNA was prepared, usingwhich as a template the genes encoding the light chain and the heavychain of the antibody was amplified. By translating the nucleotidesequence of the amplified genes, the respective amino acid sequences ofthe light chain and heavy chain variable regions were determined for theanti-hTfR antibody No. 3 produced by the cell line.

The anti-hTfR antibody No.3 was found to include the amino acid sequenceset forth as SEQ ID NO:48 as the light chain variable region, and theamino acid sequence set forth as SEQ ID NO:49 as the heavy chainvariable region. The light chain variable region was found to includethe amino acid sequence set forth as SEQ ID NO:6 or 7 as CDR1; SEQ IDNO:8 or 9 as CDR2, and SEQ ID NO:10 as CDR3; and the heavy chainvariable region to include the amino acid sequence set forth as SEQ IDNO:11 or 12 as CDR1, SEQ ID NO:13 or 14 as CDR2, and SEQ ID NO:15 or 16as CDR3. However, it was also considered that CDRs are not limited tothose which consist of these amino acid sequences, but they can alsoeither be regions of amino acid sequences that include any of the abovesequences, or amino acid sequences consisting of not less than threeconsecutive amino acids containing part of the above sequences.

Table 1 shows collectively the SEQ ID NOs of the respective amino acidsequences contained in CDR1 to CDR3 of the light chain variable regionand CDR1 to CDR3 of the heavy chain variable region of anti-hTfRantibody No. 3. However, Table 1 shows those amino acid sequence only asexamples and does not limit the amino acid sequence of each CDR to thosein Table 1, but it was considered that CDRs are not limited to thosewhich consist of these amino acid sequences, but they can also either beregions of amino acid sequences that include any of the above sequences,or amino acid sequences consisting of not less than three consecutiveamino acids containing part of the above sequences.

TABLE 1 Sequence numbers of respective amino acid sequences contained inCDR1 to CDR3 of the light chain and the heavy chain variable regions ofanti-hTfR antibodies Nos. 3 Antibody light chain variable region heavychain variable region No. CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 3 6, 7 8, 9 1011, 12 13, 14 15, 16

Example 7 Measurement of the Affinity of Anti-hTfR Antibody to Human andMonkey TfRs

The affinity of the anti-hTfR antibody to human and monkey TfRs weremeasured on Octet RED96 (ForteBio Inc., a division of Pall Corporation),a system for analysis of interactions between biomolecules utilizingbio-layer interferometry (BLI). The basic principles of bio-layerinterferometry are briefly explained below. When a layer of abiomolecule immobilized on the surface of a sensor tip is irradiatedwith light of a certain wavelength, the light is reflected from two ofthe surfaces, the one of the biomolecule and the other of inner,reference layer, producing interfering light waves. A molecule in thesample being measured binds to the biomolecule on the surface of thesensor tip and thus increases the thickness of the layers on the sensortip, which results in a shift between the interfering waves. Bymeasuring the variations of this shift between the interfering waves,determination of the number of the molecules bound to the layer of thebiomolecules immobilized to the sensor tip surface and kinetic analysisof it can be performed in real time. The measurement was performedaccording generally to the operating manual attached to Octet RED96. Asa human TfR, a recombinant human TfR (r human TfR: Sino Biological Inc.)was used, which had the amino acid sequence of the hTfR extracellularregion, i.e., the cysteine residue at position 89 from the N-terminalside to the phenylalanine at the C-terminus, of the amino acid sequenceset forth as SEQ ID NO:1, with a histidine tag attached to theN-terminus. As a monkey TfR, a recombinant monkey TfR (r monkey TfR:Sino Biological Inc.) was used, which had the amino acid sequence of thecrab-eating monkey TfR extracellular region, i.e., the cysteine residueat position 89 from the N-terminal side to the phenylalanine at theC-terminus, of the amino acid sequence set forth as SEQ ID NO:2, with ahistidine tag attached to the N-terminus.

Clone 3 line selected in Example 5 was diluted with a RPMI 1640 mediumcontaining (1×) HAT Supplement (Life Technologies Inc.) and 10% Ultralow IgG fetal bovine serum (Life Technologies Inc.) so as to adjust thecell density to approximately 2×10⁵ cells/mL. To a 1-L conical flaskwere added 200 mL of each cell suspension, and the culture was performedfor 6 to 7 days in a wet environment at 37° C., 5% CO₂ and 95% air, withstirring at a rate of about 70 rpm. The culture medium was centrifuged,and then filtered through a 0.22 μm filter (Millipore Inc.) to collect aculture supernatant. The culture supernatant thus collected was loadedonto a Protein G column (column volume: 1 mL, GE Healthcare Inc.) thathad been equilibrated in advance with three column volumes of 20 mM Trisbuffer (pH 8.0) containing 150 mM NaCl. After the column was washed with5 column volumes of the same buffer, adsorbed antibody was eluted with 4column volumes of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl,and eluted fractions were collected. The fractions eluted were adjustedto pH 7.0 by addition of 1 M Tris buffer (pH 8.0). These were used aspurified products of anti-hTfR antibody No. 3 in the experimentsdescribed below.

The purified product of anti-hTfR antibody No. 3 was subjected to 2-folddilution steps with HBS-P+ (10 mM HEPES containing 150 mM NaCl, 50 μMEDTA and 0.05% Surfactant P20) to prepare antibody solutions of 7different concentrations, 0.78125 to 50 nM (0.117 to 7.5 μg/mL). Theantibody solution was used as the sample solution. The r human and rmonkey TfRs were respectively diluted with HBS-P+ to prepare 25 μg/mLsolutions, which were used as r human TfR-ECD (Histag) solution and rmonkey TfR-ECD (Histag) solution, respectively.

Each of the sample solutions prepared above by 2-fold dilution steps wasadded, 200 μL/well, to a 96-well plate, black (Greiner Bio-One Inc.).Each of the r human TfR-ECD (Histag) solution and the r monkey TfR-ECD(Histag) solutions prepared above was added, 200 μL/well, topredetermined wells. To respective wells for baseline, dissociation andwashing were added HBS-P+, 200 μL/well. To wells for regeneration wereadded 10 mM Glycine-HCl (pH 1.7), 200 μL/well. To wells for activationwas added 0.5 mM NiCl₂ solution, 200 μL/well. The plate and biosensor(Biosensor/Ni-NTA: ForteBio Inc., a division of Pall Corporation) wereset in the prescribed positions of Octet RED96.

Octet RED96 was run under the conditions shown in Table 2 below tocollect data, on which then, using the analyzing software attached toOctet RED96, and fitting the binding reaction curve to 1:1 binding modelor 2:1 binding model, the association rate constant (kon) anddissociation rate constant (koff) of anti-hTfR antibody to r human TfRand r monkey TfR were measured and the dissociation constant (K_(D)) wascalculated. The measurement was performed at 25 to 30° C.

TABLE 2 Operating conditions of Octet RED96 Contact time Step (sec) Rate(rpm) Threshold 1 Baseline 1 60 1000 — 2 Load 600 1000 1.5-2.0 3Baseline 2 60 1000 — 4 Association 180 1000 — 5 Dissociation 540 1000 —6 Regeneration 5 1000 — 7 Washing 5 1000 — Steps 6-7 repeated 6 to 7times 8 Activation 60 1000 — Steps 1-8 repeated until all the samplesmeasured

Table 3 shows the results of measurement of association rate constant(kon), dissociation rate constant (koff) of anti-hTfR antibody No. 3,and dissociation constant (k_(D)) to human TfR and monkey TfR.

TABLE 3 Affinity of anti-hTfR antibody No. 3 for human TfR and monkeyTfR kon (M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) Human TfR 6.53 × 10⁵ <1.0 × 10⁻⁷<1.0 × 10⁻¹² Monkey TfR 3.89 × 10⁵ <1.0 × 10⁻⁷ <1.0 × 10⁻¹²

As a result of the affinity measurement of the anti-hTfR antibody tohuman TfR, the dissociation constant of anti-hTfR antibody No. 3 withhuman TfR was not more than 1×10⁻¹² M, and the dissociation constantwith monkey TfR was not more than 1×10⁻¹² M. The result shows thatanti-hTfR antibody No. 3 is an antibody having a high affinity not onlyto human TfR but also to monkey TfR.

Example 7-2 Evaluation of Transfer of the Anti-hTfR Antibodies into theBrain Using Mice

Then, for anti-hTfR antibody No. 3, evaluation was performed about thetransfer into the brain through the BBB, by using hTfR knock-in mice(hTfR-KI mice) in which the gene encoding the extracellular region ofmouse transferrin receptor has been replaced with a gene encoding theextracellular region of human transferrin receptor. The hTfR-KI micewere produced by the method described below as a whole. Besides, thepurified products prepared in Example 7 were used as anti-hTfR antibodyNo. 3.

A DNA fragment having a nucleotide sequence set forth as SEQ ID NO:45was chemically synthesized, in which a neomycin resistance gene flankedby loxP sequences was placed on the 3′-side of a cDNA encoding achimeric hTfR whose intracellular region consisted of the amino acidsequence of mouse TfR and the extracellular region consisted of theamino acid sequence of human hTfR sequence. This DNA fragment wasinserted by a conventional method into a targeting vector having as the5′-arm sequence a nucleotide sequence set forth as SEQ ID NO:46 and asthe 3′-arm sequence a nucleotide sequence set forth as SEQ ID NO:47, andthe construct was introduced into mouse ES cells by electroporation. Themouse ES cells to which the gene had been introduced were subjected toselection culture in a medium in the presence of neomycin to selectthose mouse ES cells in which the targeting vector had been incorporatedinto the chromosome through homologous recombination. The recombinantmouse ES cells thus obtained were injected into 8-cell stage embryos(host embryos) of ICR mice, and the embryos thus prepared were implantedinto pseudo pregnant mice (recipient mice) which had been obtainedthrough mating with mice having undergone vasoligation. The offspring(chimeric mice) obtained were examined by their hair color, and thosemice which had the higher proportion of white hairs in their total bodyhairs were selected, i.e., those mice in which the ES cells hadcontributed at the higher rates in the development of the individualorganisms. Each of these chimeric mice was mated with ICR mice togenerate F1 mice. F1 mice with white hair were selected, the DNAsextracted from their tail tissue were analyzed, and those mice whosemouse transferrin receptor gene on their chromosomes had been replacedwith chimeric hTfR, were regarded as hTfR-KI mice.

The purified product of anti-hTfR antibody No. 3 were fluorescentlabeled with fluorescein isothiocyanate (FITC) using FluoresceinLabeling Kit-NH₂ (Dojindo Laboratories) according to the attachedmanual. PBS solution containing the FITC fluorescent labeled antibodywas prepared. The PBS antibody solution was intravenously injected to anhTfR-KI mouse (male, 10 to 12-week old), at the anti-hTfR antibodydosage of 3 mg/kg. As a control, a PBS solution containing mouse IgG1(Sigma Inc.), fluorescent labeled with FITC in the same manner as above,was intravenously injected to an hTfR-KI mouse (male, 10 to 12-weekold), at the dose of 3 mg/kg. About eight hours after the intravenousinjection, the whole body was irrigated with physiological saline, andthe brain (part including the cerebrum and the cerebellum) obtained. Thebrain thus excised was weighed (wet weight), and then the brain tissueswere homogenized with T-PER (Thermo Fisher Scientific Inc.) containingProtease Inhibitor Cocktail (Sigma Inc.). The homogenate wascentrifuged, the supernatant was collected, and the amount of the FITCfluorescent labeled antibody contained in the supernatant was measuredin the following manner. First, 10 μL of anti-FITC Antibody (BethylInc.) was added to each well of a High Bind Plate (Meso ScaleDiagnostics Inc.) and left to stand for one hour so as to immobilize itto the plate. Then, the plate was blocked by addition of 150 μL ofSuperBlock Blocking buffer in PBS (Thermo Fisher Scientific Inc.) toeach well and shaking of the plate for one hour. Then, 25 μL of thesupernatant of a brain tissue homogenate was added to each well, and theplate was shaken for one hour. Then, 25 μL of SULFO-TAG Anti-MouseAntibody(Goat)(Meso Scale Diagnostics Inc.) were added to each well, andshaking was continued for one hour. Then, to each well was added 150 μLof Read buffer T (Meso Scale Diagnostics Inc.), and the amount ofluminescence from each well was read on a Sector™ Imager 6000 reader.The amount of the anti-hTfR antibody contained per one gram brain (wetweight) (the concentration of the anti-hTfR antibody in the braintissues) was calculated, by producing a standard curve based onmeasurements of standard samples containing known concentrations of FITCfluorescent labeled anti-hTfR antibody, and then interpolating themeasurement of each of the samples with reference to the standard. Theresults are shown in Table 4.

The concentration of anti-hTfR antibody No. 3 in brain tissues was about27.8 times as high as that of the control. The result indicates thatanti-hTfR antibody No. 3 transfers into the brain, actively passingthrough the BBB.

TABLE 4 Concentration of anti-hTfR antibodies in in brain tissuesAntibody Brain tissues No. (μg/g wet weight) Relative value to thecontrol Control 0.003 1 3 0.0833 27.8

Example 8 Pharmacokinetic Analysis of Anti-hTfR Antibodies in Monkey

Anti-hTfR antibody No. 3 was intravenously administered once to a malecrab-eating monkey at a dosage of 5.0 mg/kg, and 8 hours after theadministration, whole body irrigation was carried out with physiologicalsaline As a negative control, a monkey which had not received anti-hTfRantibody was subjected to whole body irrigation in the same manner.After the irrigation, brain tissues including the medulla oblongata wereexcised. Using the brain tissues, the concentration of the anti-hTfRantibody was measured, and immunohistochemical staining was performed.Anti-hTfR antibody No. 3 employed were purification products of thosedescribed in Example 7.

Measurement of the concentration of anti-hTfR antibodies in braintissues were carried out largely following the procedure describedbelow. Collected brain tissues were divided into the cerebrum, thecerebellum, the hippocampus, and the medulla oblongata, and they wererespectively homogenized with RIPA Buffer (Wako Pure Chemical IndustriesInc.) containing Protease Inhibitor Cocktail (Sigma-Aldrich Inc.), andcentrifuged to collect the supernatant. Affinipure Goat Anti mouse IgGFcγ pAb (Jackson ImmunoResearch Inc.) was added, 10 μL each, to thewells of a High Bind Plate (Meso Scale Diagnostics Inc.), and the platewas left to stand for one hour to immobilize the antibody. Then, theplate was blocked by addition of 150 μL of SuperBlock Blocking buffer inPBS (Thermo Fisher Scientific Inc.) to each well and shaken for onehour. Then, 25 μL of the supernatant of a brain tissue homogenate wasadded to each well, and the plate was shaken for one hour. Then, 25 μLof Affinipure Goat Anti mouse IgG Fab-Biotin (Jackson ImmunoResearchInc.) was added to each well, and shaking was continued for one hour.Then, 25 μL or SULFO-Tag-Streptavidin (Meso Scale Diagnostics Inc.) wasadded to each well, and shaking was continued for 30 minutes. To eachwell was added 150 μL of Read buffer T (Meso Scale Diagnostics Inc.),and the amount of luminescence from each well was read on a Sector™Imager 6000 reader (Meso Scale Diagnostics). The amount of the anti-hTfRantibody contained per one gram of brain (wet weight) (the concentrationof the anti-hTfR antibody in brain tissues) was calculated, by producinga standard curve based on measurements of standard samples containingknown concentrations of the anti-hTfR antibody, and then interpolatingthe measurement of each of the samples with reference to the standard.

The result of the measurement of the concentration of the anti-hTfRantibodies in brain tissues is shown in Table 5. Anti-hTfR antibody No.3 was observed to accumulate in all of the cerebrum, the cerebellum, thehippocampus and the medulla oblongata. These results demonstrate thatanti-hTfR antibody No. 3 had a property to pass through the blood brainbarrier and accumulate in the brain tissues, and show that by bindingthese antibodies to a pharmaceutical agent which needs to be broughtinto function in the brain tissues, it is possible to let thosepharmaceutical agents efficiently accumulate in the brain tissues.

TABLE 5 Concentration of anti-hTfR antibodies in brain tissues (μg/g wetweight) Antibody Hippo- Cervical No. Cerebrum Cerebellum campus cord 30.72 0.6 0.33 0.31

Immunohistochemical staining of the anti-hTfR antibodies in these braintissues was carried out following the procedures described below as awhole. The collected tissues were rapidly frozen to −80° C. in aTissue-Tek Cryo 3DM (Sakura Finetek Inc.) to prepare frozen blocks oftissues. The frozen blocks were sliced into 4-μm sections, and whichwere applied to MAS coated glass slides (Matsunami Glass Inc.). Thetissue sections were reacted with 4% paraformaldehyde (Wako PureChemical Industries Inc.) for 5 minutes at 4° C. and affixed to glassslides. Then, the tissue sections were reacted with methanol solutioncontaining 0.3% hydrogen peroxide (Wako Pure Chemical Industries Inc.)for 30 min to inactivate intrinsic peroxidases. Then, the glass slideswere blocked by reacting SuperBlock blocking buffer in PBS for 30 min atroom temperature. Then, the tissue sections were reacted with MouseIgG-heavy and light chain Antibody (Bethyl Laboratories Inc.) for onehour at room temperature. The tissue sections were allowed to develop acolor with DAB substrate (3,3′-diaminobenzidine, Vector LaboratoriesInc.), counterstained with Mayer's hematoxylin solution (Merck Inc.),dehydrated, cleared, embedded, and observed under a microscope.

FIG. 1 shows the result of the immunohistochemical staining of theanti-hTfR antibodies in the cerebral cortex. In the cerebral cortex ofmonkeys administered anti-hTfR antibody No. 3, specific staining ofblood vessels were observed (FIG. 1b ). Further, the brain parenchymaregion, outside the blood vessels, was also observed specificallystained extensively. In contrast, no staining was observed in thecerebral cortex of the control monkey administered with no anti-hTfRantibody, indicating that there was almost no background staining (FIG.1a ).

FIG. 2 shows the result of immunohistochemical staining of anti-hTfRantibodies in the hippocampus. In the cerebral cortex of monkeysadministered anti-hTfR antibody No. 3, specific staining of bloodvessels was observed (FIG. 2b ). Further, specific staining ofnerve-like cells was also observed, and specific and extensive stainingof the brain parenchyma region, outside the blood vessels, was alsoobserved. On the other hand, no staining was observed in the hippocampusof the control administered with no anti-hTfR antibody, indicating thatthere was almost no background staining (FIG. 2a ).

FIG. 3 shows the result of immunohistochemical staining of the anti-hTfRantibodies in the cerebellum. In the cerebral cortex of monkeysadministered anti-hTfR antibody No. 3, specific staining of bloodvessels were observed (FIG. 3b ). Further, specific staining of Purkinjecells was also observed. In contrast, no staining was observed in thecerebellum of the control with no anti-hTfR antibody administered,indicating that there was almost no background staining (FIG. 3a ).

From the above results of immunohistochemical staining in the cerebrum,the hippocampus, and the cerebellum, it was found that anti-hTfRantibody No. 3 can bind to hTfR occurring on the endothelium of bloodvessels of the brain, and after binding to hTfR, they pass through theblood-brain barrier and transfer into the brain parenchyma, and further,from the brain parenchyma to nerve-like cells in the hippocampus, andare taken up by Purkinje cells in the cerebellum.

Example 9 Preparation of Humanized Anti-hTfR Antibodies

Humanization was tried of the amino acid sequence included in the lightchain and the heavy chain variable regions of anti-hTfR antibody No. 3.Thus, a humanized light chain variable region having one of the aminoacid sequences set forth as SEQ ID NO:17 to SEQ ID NO:22, and ahumanized heavy chain variable region having one of the amino acidsequences set forth as SEQ ID NO:31 to SEQ ID NO:36 were obtained.

Example 10 Construction of Genes Encoding Humanized Anti-hTfR Antibodies

For anti-hTfR antibody No. 3 above, DNA fragments were artificiallysynthesized which contained a gene encoding the full length of the lightchain, and of the heavy chain, having humanized anti-hTfR antibody lightchain and heavy chain variable regions, respectively. In doing this, aMluI sequences and a sequence encoding a leader peptide was added, inthis order from the 5′ end, on the 5′ side of the gene encoding the fulllength of the light chain, and on the 3′ side was added a NotI sequence.And, a MluI sequences and a sequence encoding a leader peptide wasadded, in this order from the 5′ end, on the 5′ side of the geneencoding the full length of the heavy chain, and on the 3′ side wasadded a NotI sequence. The leader peptide introduced above is tofunction as secretion signal when the light chain and heavy chain of thehumanized antibody is expressed in mammalian cells as host cells so thatthe light chain and the heavy chain are secreted out of the cells.

For the light chain of anti-hTfR antibody No.3, a DNA fragment (SEQ IDNO:24) was synthesized, which included a gene encoding the full lengthof the light chain (the light chain of humanized anti-hTfR antibodyNo.3) consisting of the amino acid sequence set forth as SEQ ID NO:23,which had in the variable region the amino acid sequence set forth asSEQ ID NO:18.

As to the light chain of anti-hTfR antibody No.3, also synthesized were,

a DNA fragment (SEQ ID NO:26) encoding the full length amino acidsequence of the light chain (the light chain of humanized anti-hTfRantibody No.3-2) consisting of the amino acid sequence set forth as SEQID NO:25, which had in the variable region the amino acid sequence setforth as SEQ ID NO:20;

a DNA fragment (SEQ ID NO:28) encoding the full length amino acidsequence of the light chain (the light chain of humanized anti-hTfRantibody No.3-3) consisting of the amino acid sequence set forth as SEQID NO:27, which had in the variable region the amino acid sequence setforth as SEQ ID NO:21;

a DNA fragment (SEQ ID NO:30) encoding the full length amino acidsequence of the light chain (the light chain of humanized anti-hTfRantibody No.3-4) consisting of the amino acid sequence set forth as SEQID NO:29, which had in the variable region the amino acid sequence setforth as SEQ ID NO:22.

For the heavy chain of anti-hTfR antibody No. 3, a DNA fragment (SEQ IDNO: 38) was synthesized, which encoded the full length of the heavychain (the heavy chain of humanized anti-hTfR antibody No. 3) consistingof the amino acid sequence set forth as SEQ ID NO: 37, which had in thevariable region the amino acid sequence set forth as SEQ ID NO: 32. Theheavy chain of the humanized anti-hTfR antibody encoded by the DNAfragment set forth as SEQ ID NO: 38 is IgG1.

Further, for the heavy chain of anti-hTfR antibody No.3, alsosynthesized was a DNA fragment (SEQ ID NO:40) encoding the full lengthamino acid sequence of the heavy chain (the heavy chain IgG4 ofhumanized anti-hTfR antibody No.3) consisting of the amino acid sequenceset forth as SEQ NO:39, which had in the variable region the amino acidsequence set forth as SEQ ID NO:32;

The heavy chain of the humanized anti-hTfR antibody encoded by the DNAfragment set forth as SEQ ID NO:40 is IgG4.

Example 11 Construction of Humanized Anti-hTfR Antibody ExpressionVector

Vector pEF/myc/nuc (Invitrogen Inc.) was digested with KpnI and NcoI tocut out a region including EF-1α promoter and its first intron, and thiswas blunt-ended with T4 DNA polymerase. A region including the CMVenhancer/promoter and intron was removed from pCI-neo (Invitrogen Inc.)by digesting it with BglII and EcoRI, and the remaining fragment thusleft was blunt-ended with T4 DNA polymerase. To this was inserted theabove-mentioned region including EF-1α promoter and its first intron toconstruct pE-neo vector. This vector, pE-neo, was digested with SfiI andBstXI to remove a region of approximately 1 kb including a neomycinresistance gene. PCR was performed employingpcDNA3.1/Hygro(+)(Invitrogen) as a template and using primer Hyg-Sfi5′(SEQ ID NO:43) and primer Hyg-BstX3′ (SEQ ID NO:44) to amplifyhygromycin gene. The hygromycin gene thus amplified was digested withSfiI and BstXI and inserted into the above pE-neo vector from whichneomycin resistance gene had been removed to construct a vector pE-hygr.

Vectors pE-hygr and pE-neo were both digested with MluI and NotI. TheDNA fragment (SEQ ID NO:24) encoding the light chain of humanizedanti-hTfR antibody No. 3 and the DNA fragment (SEQ ID NO:38) encodingthe heavy chain of the antibody, both synthesized in Example 10, weredigested with MluI and NotI, and the fragments thus obtained wereinserted into vector pE-hygr and vector pE-neo, respectively, betweentheir MluI and NotI sites. The vectors thus obtained were used as anexpression vector for the light chain of humanized anti-hTfR antibodyNo. 3, pE-hygr(LC3), and as an expression vector for the heavy chain ofthe antibody, pE-neo(HC3), in the experiments described below.

Further, as to the light chain of anti-hTfR antibody No.3, the followingfragments synthesized in Example 10, namely:

the DNA fragment (SEQ ID NO:26) encoding the light chain of humanizedanti-hTfR antibody No.3-2,

the DNA fragment (SEQ ID NO:28) encoding the light chain of humanizedanti-hTfR antibody No.3-3, and

the DNA fragment (SEQ ID NO:30) encoding the light chain of humanizedanti-hTfR antibody No.3-4,

were digested with MluI and NotI, and inserted into the vector pE-hygrbetween the MluI and NotI sites thereof to construct

pE-hygr(LC3-2), an expression vector for the light chain of humanizedanti-hTfR antibody No.3-2,

pE-hygr(LC3-3), an expression vector for the light chain of humanizedanti-hTfR antibody No.3-3, and

pE-hygr(LC3-4), an expression vector for the light chain of humanizedanti-hTfR antibody No.3-4, respectively.

Further, in the same manner as above, as to the heavy chain of anti-hTfRantibody No. 3, the DNA fragment (SEQ ID NO:40) encoding the heavy chainIgG4 of humanized anti-hTfR antibody No.3 synthesized in Example 10 wasdigested with MluI and NotI, and inserted into the vector pE-neo betweenthe MluI and NotI sites thereof to construct pE-neo(HC3-IgG4), anexpression vector for the heavy chain IgG4 of humanized anti-hTfRantibody No. 3.

Example 12 Construction of Cells for Expression of Humanized Anti-hTfRAntibody

CHO cells (CHO-K1: purchased from American Type Culture Collection) weretransformed with pE-hygr(LC3), the vector for light chain expression,and pE-neo(HC3), the vector for heavy chain expression, both constructedin Example 11, as follows, using GenePulser (Bio-Rad Inc.).Transformation of the cells was performed in the following manner as awhole. CHO-K1 cells, 5×10⁵, were seeded in a 3.5-cm culture dishcontaining CD OptiCHO™ medium (Life Technologies Inc.) and culturedovernight at 37° C., 5% CO₂. The medium was replaced with Opti-MEM™ Imedium (Life Technologies Inc.), and the cells were suspended at thedensity of 5×10⁶ cells/mL. One hundred μL of the cell suspension weretaken, to which was added 5 μL each of a pE-hygr(LC3) solution and apE-neo(HC3) plasmid DNA solution both having been diluted with Opti-MEM™I medium to 100 μg/mL. These plasmids were introduced into the cells byelectroporation using GenePulser (Bio-Rad Inc.). The cells then werecultured overnight under the condition of 37° C., 5% CO₂, and subjectedto selection culture in D OptiCHO™ medium supplemented with 0.5 mg/mL ofhygromycin and 0.8 mg/mL of G418.

Then, the cells selected above through the selection culture were seededon 96-well plates so that not more than one cell was seeded per well bylimiting dilution. The cells then were cultured for about 10 days sothat monoclonal colonies were formed. Respective culture supernatants ofthe wells in which monoclonal colony was formed were collected, theamount of the humanized antibody contained in culture supernatants wasdetermined by ELISA, and humanized antibody high-expressing cell lineswere selected.

The ELISA above was conducted as follows in general. To each well of96-well microtiter plates (Nunc Inc.) were added 100 μL of a goatanti-human IgG polyclonal antibody solution diluted with 0.05 M sodiumbicarbonate buffer (pH 9.6) to 4 μg/mL, and the plate was left to standfor at least one hour at room temperature so as to allow the antibody tobe adsorbed by the plates. Then, after the well were washed three timeswith PBS-T, 200 μL of Starting Block (PBS) Blocking Buffer (ThermoFisher Scientific Inc.) was added to each well, and the plates were leftto stand for 30 minutes at room temperature. After each well was washedwith PBS-T three times, the culture supernatant or the human IgGstandard product which had been diluted with a PBS supplemented with0.5% BSA and 0.05% Tween20 (PBS-BT) to appropriate concentrations, wasadded to each well, in the amount of 100 μL, and the plates were left tostand for at least one hour at room temperature. After the plates werewashed three times with PBS-T, 100 μL of HRP-labeled anti-human IgGpolyclonal antibody solution which had been diluted with PBS-BT, wasadded to each well, and the plates were left to stand for at least onehour at room temperature. After the wells were washed three times withPBS-T, 0.4 mg/mL o-phenylenediamine in citrate-phosphate buffer (pH 5.0)was added to each well, in the amount of 100 μL, and the wells were leftto stand for 8 to 20 minutes at room temperature. Then, 1 mol/L sulfuricacid was added to each well, in the amount of 100 μL to terminate thereaction, and the absorbance for each well was measured at 490 nm usinga 96-well plate reader. The cells corresponding to the wells whichexhibited the higher measurements were regarded as a high-expressingcell line for humanized anti-hTfR antibody No. 1. This was designatedantibody No.3 expressing cell line.

Further, in the same manner, CHO cells were transformed with the lightchain expression vector pE-hygr(LC3-2) and the heavy chain expressionvector pE-neo(HC3), both constructed in Example 11, and ahigh-expressing cell line for humanized anti-hTfR antibody No.3-2 wasobtained. This was designated antibody No.3-2 expressing cell line.

Further, in the same manner, CHO cells were transformed with the lightchain expression vector pE-hygr(LC3-3) and the heavy chain expressionvector pE-neo(HC3), both constructed in Example 11, and ahigh-expressing cell line for humanized anti-hTfR antibody No.3-3 wasobtained. This was designated antibody No.3-3 expressing cell line.

Further, in the same manner, CHO cells were transformed with the lightchain expression vector pE-hygr(LC3-4) and the heavy chain expressionvector pE-neo(HC3) both constructed in Example 11, and a high-expressingcell line for humanized anti-hTfR antibody No.3-4 was obtained. This wasdesignated antibody No.3-4 expressing cell line.

Further, in the same manner, CHO cells were transformed with the lightchain expression vector pE-hygr(LC3) and the heavy chain expressionvector pE-neo(HC3-IgG4) both constructed in Example 11, and ahigh-expressing cell line for humanized anti-hTfR antibody No.3(IgG4)was obtained. This was designated antibody No.3(IgG4) expressing cellline.

Further, in the same manner, CHO cells were transformed with the lightchain expression vector pE-hygr(LC3-2) and the heavy chain expressionvector pE-neo(HC3-IgG4) both constructed in Example 11, and ahigh-expressing cell line for humanized anti-hTfR antibody No.3-2 (IgG4)was obtained. This was designated antibody No.3-2 (IgG4) expressing cellline.

Example 13 Purification of Humanized Anti-hTfR Antibodies

Antibody No.3 expressing cell line, antibody No.3-2 expressing cellline, antibody No.3-3 expressing cell line and antibody No.3-4expressing cell line obtained in Example 12 were respectively dilutedwith CD OptiCHO™ medium to the density of approximately 2×10⁵ cells/mL.The cell suspensions, 200 mL, was added to a 1 L-conical flask, andcultured for 6 to 7 days in a wet environment at 37° C., 5% CO₂, 95%air, with stirring at a rate of about 70 rpm. Each culture supernatantwas collected by centrifugation, and filtered through a 0.22 μm filter(Millipore Inc.) to prepare the culture supernatant. To each culturesupernatant thus obtained was added five volumes of 20 mM Tris buffer(pH 8.0) containing 150 mM NaCl, and loaded onto a Protein A column(column volume: 1 mL, Bio-Rad Inc.) which had been equilibrated inadvance with three column volumes of 20 mM Tris buffer (pH 8.0)containing 150 mM NaCl. Then, the column was washed with five columnvolumes of the same buffer, and the adsorbed humanized antibody waseluted with four column volumes of 50 mM glycine buffer (pH 2.8)containing 150 mM NaCl, and the eluted fraction was collected. Theeluted fractions was added and neutralized with 1 M Tris buffer (pH 8.0)and used as the purified antibody preparation.

In the above, the antibody purified from the culture supernatant ofantibody No.3 expressing cell line was designated humanized anti-hTfRantibody No.3. The antibody purified from the culture supernatant ofantibody No.3-2 expressing cell line was designated humanized anti-hTfRantibody No.3-2. The antibody purified from the culture supernatant ofantibody No.3-3 expressing cell line was designated humanized anti-hTfRantibody No.3-3. The antibody purified from the culture supernatant ofantibody No.3-4 expressing cell line was designated humanized anti-hTfRantibody No.3-4.

Further, antibody No.3(IgG4) expressing cell line and antibody No.3-2(IgG4) expressing cell line obtained in Example 12 also were cultured inthe same manner as above, and from their culture supernatants wereobtained purified humanized anti-hTfR antibody No.3(IgG4) and humanizedanti-hTfR antibody No.3-2 (IgG4), respectively. These two antibodieswere employed in the pharmacokinetic analysis using monkeys described inExample 15.

Example 14 Measurement of Affinity of Humanized Anti-hTfR Antibodies toHuman TfR and Monkey TfR

The affinity of the humanized anti-hTfR antibodies obtained in Example13 to human and monkey TfRs was measured by the method described inExample 7. Table 6 shows the result of the measurement of theassociation rate constant (kon), dissociation rate constant (koff), anddissociation constant (k_(D)) of humanized anti-hTfR antibodies Nos. 3to 3-4 (corresponding to Nos. 3 to 3-4, respectively, in the table) tohuman TfR.

TABLE 6 Affinity of humanized anti-hTfR antibodies to human TfR AntibodyNo. kon (M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) 3 1.19 × 10⁶ <1.0 × 10⁻⁷ <1.0 ×10⁻¹² 3-2 6.06 × 10⁵ 1.45 × 10⁻⁵ 2.39 × 10⁻¹¹ 3-3 6.00 × 10⁵ 1.25 × 10⁻⁵2.09 × 10⁻¹¹ 3-4 1.01 × 10⁶ <1.0 × 10⁻⁷ <1.0 × 10⁻¹²

Table 7 shows the result of the measurement of the association rateconstant (kon), dissociation rate constant (koff), and dissociationconstant (k_(D)) of humanized anti-hTfR antibodies Nos. 3 to 3-4(corresponding to Nos. 3 to 3-4, respectively, in the table) to monkeyTfR.

TABLE 7 Affinity of humanized anti-hTfR antibodies to monkey TfRAntibody No. kon (M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) 3 6.03 × 10⁵ 6.76 × 10⁻⁴1.12 × 10⁻⁹ 3-2 4.95 × 10⁵ 8.76 × 10⁻⁴ 1.77 × 10⁻⁹ 3-3 4.88 × 10⁵ 9.32 ×10⁻⁴ 1.91 × 10⁻⁹ 3-4 5.19 × 10⁵ 1.35 × 10⁻⁴  2.60 × 10⁻¹⁰

The result of the measurement of the affinity of humanized anti-hTfRantibody Nos. 3 to 3-4 to human TfR showed that the dissociationconstant between humanized anti-hTfR antibodies Nos. 3 and 3-4 and humanTfR was less than 1×10⁻¹² M (Table 6). And the dissociation constantbetween humanized anti-hTfR antibodies Nos. 3-2 and 3-3 and human TfRwas 2.39×10⁻¹¹ M and 2.09×10⁻¹¹ M, respectively. At the same time, thedissociation constant between the pre-humanized anti-hTfR antibodies(antibody No. 3) corresponding to those antibodies and human TfR was:1×10⁻¹² M (Table 3). These results demonstrate that the high affinity ofthose pre-humanized anti-hTfR antibodies to human TfR was maintainedafter humanization of the antibodies.

Then, looking to the result of measurement of the affinity of humanizedanti-hTfR antibodies to monkey TfR, regarding to humanized anti-hTfRantibodies Nos. 3 to 3-4, while the dissociation constant of anti-hTfRantibody No.3, the pre-humanized antibody corresponding to them, tomonkey TfR was less than 1×10⁻¹² M, their dissociation constant afterhumanization was 2.60×10⁻¹⁰ M to 1.91×10⁻⁹ M, showing a lowering of theaffinity to monkey TfR. As to the humanized anti-hTfR antibody No.3,although a lowering of affinity to monkey TfR was observed, the resultindicates that the pre-humanized high affinity of anti-hTfR antibody tomonkey TfR was not lost after its humanization but was maintained as awhole.

Example 15 Pharmacokinetic Analysis of Humanized Anti-hTfR Antibody inMonkey

Using monkeys, pharmacokinetic analysis was performed with fourantibodies: humanized anti-hTfR antibody No.3, humanized anti-hTfRantibody No.3-2, humanized anti-hTfR antibody No.3 (IgG4), and humanizedanti-hTfR antibody No.3-2 (IgG4). Besides, the heavy chain of humanizedanti-hTfR antibody No.3 was IgG1, while in humanized anti-hTfR antibodyNo.3 (IgG4), the heavy chain of humanized anti-hTfR antibody No.3 hadbeen converted into IgG4, with its variable region kept intact. Further,the heavy chain of humanized anti-hfR antibody No.3-2 was IgG1, while inhumanized anti-hTfR antibody No.3-2 (IgG4), the heavy chain of humanizedanti-hTfR antibody No.3-2 had been converted into IgG4 with its variableregion kept intact. These four antibodies were respectivelyintravenously administered once to male crab-eating monkeys, at a dosageof 5.0 mg/kg, and their peripheral blood was sampled before theadministration, 2 minutes, 30 minutes, 2 hours, 4 hours and 8 hoursafter the administration, and then they were subjected to whole bodyirrigation. As a negative control, trastuzumab (Herceptin™, ChugaiPharmaceutical Co., Ltd.), a humanized antibody to HER2 protein, wasintravenously administered once to a single monkey in the same manner,and its peripheral blood was sampled before the administration, 2minutes, 30 minutes, 2 hours, 4 hours and 8 hours after theadministration, and then it was subjected to the whole body irrigation.After the irrigation, the brain and spine tissues including the medullaoblongata and other tissues (liver, heart, spleen and bone marrow) wereexcised. Using these brain and spinal tissues and other tissues, theconcentration of the humanized anti-hTfR antibodies was measured andimmunohistochemical staining was carried out.

Measurement of the concentration of humanized anti-hTfR antibodies intissues and peripheral blood was carried out largely following theprocedure described below. Besides, as to the brain, the obtainedtissues were separated into the cerebral cortex, the cerebellum, thehippocampus and the medulla oblongata, and then the concentration of thehumanized anti-hTfR antibodies were measured. The respective tissuesthus obtained were homogenized with RIPA Buffer (Wako Pure ChemicalIndustries Inc.) containing Protease Inhibitor Cocktail (Sigma-AldrichInc.), centrifuged, and the supernatant collected. From the aboveperipheral blood, serum was separated. Anti-Human Kappa Light Chain GoatIgG Biotin (Immuno-Biological Laboratories Co, Ltd.), Sulfo-taganti-human IgG (H+L) antibody (Bethyl Inc.) and the brain tissuehomogenate were added to a streptavidin plate (Meso Scale DiagnosticsInc.) blocked with SuperBlock blocking buffer in PBS (Thermo FisherScientific Inc.), and the plate was shaken for one hour forimmobilization. To each well was added 150 μL of Read buffer T (MesoScale Diagnostics Inc.), and the amount of luminescence from each wellwas measured on a Sector™ Imager 6000 reader. The amount of the antibodycontained in each tissue and the peripheral blood was calculated byproducing a standard curve based on measurements of standard samplescontaining known concentrations of the anti-hTfR antibody, and theninterpolating the measurement of each of the samples with reference tothe standard. Measurement of concentration was repeated three times foreach sample.

The result of measurement of the concentration of humanized anti-hTfRantibodies in the brain and spinal tissues is shown in Table 9.

TABLE 8 Concentration of humanized anti-hTfR antibodies in brain tissues(μg/g wet weight) Antibody Cerebral Hippo- Medulla Spinal No. cortexCerebellum campus oblongata cord 3 0.67 ± 0.12 0.61 ± 0.02 0.49 ± 0.020.59 ± 0.10 0.46 ± 0.17 3-2 1.05 ± 0.07 0.72 ± 0.04 0.72 ± 0.07 0.69 ±0.03 0.46 ± 0.02 3(IgG4) 0.65 ± 0.05 0.59 ± 0.03 0.56 ± 0.02 0.59 ± 0.020.46 ± 0.07 3-2(IgG4) 0.76 ± 0.02 0.57 ± 0.07 0.62 ± 0.05 0.73 ± 0.160.48 ± 0.03 Negative 0.0082 ± 0.0032 0.0090 ± 0.0067 0.0053 ± 0.00090.011 ± 0.003 0.15 ± 0.04 control

All the antibodies, i.e., humanized anti-hTfR antibody No.3, humanizedanti-hTfR antibody No.3-2, humanized anti-hTfR antibody No.3 (IgG4) andhumanized anti-hTfR antibody No.3-2 (IgG4), were observed to accumulatein the cerebral cortex, cerebellum, hippocampus, medulla oblongata andspinal cord (Table 8). The respective amount accumulated was as follow:

with humanized anti-hTfR antibody No.3, approximately 82 times in thecerebral cortex, approximately 68 times in the cerebellum, approximately92 times in the hippocampus, approximately 54 times in the medullaoblongata, and approximately 3.1 times in the spinal cord, in comparisonwith the negative control, trastuzumab (Herceptin™),

with humanized anti-hTfR antibody No.3-2, approximately 128 times in thecerebral cortex, approximately 80 times in the cerebellum, approximately136 times in the hippocampus, approximately 63 times in the medullaoblongata, approximately 3.1 times in the spinal cord, in comparisonwith the negative control, trastuzumab,

with humanized anti-hTfR antibody No.3 (IgG4), approximately 79 times inthe cerebral cortex, approximately 66 times in the cerebellum,approximately 106 times in the hippocampus, approximately 54 times inthe medulla oblongata, approximately 3.1 times in the spinal cord, incomparison with the negative control, trastuzumab, and

with humanized anti-hTfR antibody No.3-2 (IgG4), approximately 93 timesin the cerebral cortex, approximately 63 times in the cerebellum,approximately 117 times in the hippocampus, approximately 66 times inthe medulla oblongata, approximately 3.2 times in the spinal cord, incomparison with the negative control, trastuzumab (Table 9).

These results indicate that these four humanized anti-hTfR antibodieshave a property that allows them to pass through the blood-brain barrierand accumulate in the brain tissues, and that it is now possible to letpharmaceutical agents which need to be brought into function in thebrain tissues efficiently accumulate there, by binding suchpharmaceutical agents to one of these antibodies.

TABLE 9 Amount of humanized anti-hTfR antibodies accumulated in braintissues (factors in comparison with negative control) Antibody CerebralHippo- Medulla Spinal No. cortex Cerebellum campus oblongata cord 3 8268 92 54 3.1 3-2 128 80 136 63 3.1 3(IgG4) 79 66 106 54 3.1 3-2(IgG4) 9363 117 66 3.2 Negative 1 1 1 1 1 control

Then, FIG. 4 shows the result of measurement of the concentration of thehumanized anti-hTfR antibodies in the tissues of the liver, heart,spleen and bone marrow. The four humanized anti-hTfR antibodies, as wellas the negative control, trastuzumab, were observed to accumulate in theliver and spleen, and their amount accumulated was equal between thefour humanized anti-hTfR antibodies and trastuzumab. In the heart, thehumanized anti-hTfR antibodies tended to accumulate more thantrastuzumab, the negative control, but the amount was only about 1.5 to2.8 times that of the negative control. In bone marrow, the humanizedanti-hTfR antibodies tended to accumulate markedly more thantrastuzumab, the negative control, and the amount was 3.5 to 16 timesthat of the negative control. The cause of this accumulation of thehumanized anti-hTfR antibodies in bone marrow is thought to be that TfRis expressed at high levels in bone marrow, hematopoietic organ, andmore humanized anti-hTfR antibodies, therefore, accumulate throughbinding to TfR, than the negative control. These data indicate that thefour humanized anti-hTfR antibodies has a property that allows them tospecifically accumulate the cerebrum, cerebellum, hippocampus andmedulla oblongata, which constitute the central nervous system, and thatit is now possible to let pharmaceutical agents which need to be broughtinto function in the brain tissues efficiently accumulate there, bybinding such pharmaceutical agents to one of these antibodies.

Then, Table 9-2 shows the result of pharmacokinetic measurement of thehumanized anti-hTfR antibodies in the blood. As that of the negativecontrol, trastuzumab, the blood concentration of the four humanizedanti-hTfR antibodies was maintained at high levels, higher than 60μg/mL, even eight hours after administration, indicating that they arestable in the blood (Table 9-2).

TABLE 9-2 Pharmacokinetics of humanized anti-hTfR antibodies in blood(μg/mL blood ) Antibody Time after administration No. 2 min 30 min 2 hr4 hr 8 hr 3 173 147 128 117 97.5 3-2 124 99.5 78.5 76.5 61 3(IgG4) 141113 99 95 83 3-2(IgG4) 132 111 98.5 99 95.5 Negative 124 92.5 96 75.560.5 control

Immunohistochemical staining of the humanized anti-hTfR antibodies inbrain tissues was performed by the method described in Example 8.However, in doing this, Human IgG-heavy and light chain Antibody (BethylLaboratories Inc.) was used in place of Mouse IgG-heavy and light chainAntibody (Bethyl Laboratories Inc.).

FIG. 5 shows the result of immunohistochemical staining of the humanizedanti-hTfR antibodies in the cerebral cortex. Specific staining of bloodvessels and nerve-like cells were observed in the cerebral cortex of themonkeys administered with humanized anti-hTfR antibody No.3, humanizedanti-hTfR antibody No.3-2, humanized anti-hTfR antibody No.3 (IgG4), andhumanized anti-hTfR antibody No.3-2 (IgG4) (FIG. 5b -e, respectively).In the cerebral cortex of the monkey administered with humanizedanti-hTfR antibody No.3-2, in particular, (FIG. 5c ), the brainparenchyma region, outside the blood vessels, was also observedspecifically stained extensively. Besides, no staining was observed inthe cerebral cortex of the monkey administered with Herceptin as acontrol, indicating that the tissue staining observed in FIG. 5b-e wasspecific for the humanized anti-hTfR antibodies (FIG. 5a ).

FIG. 6 shows the result of immunohistochemical staining of the humanizedanti-hTfR antibodies in the hippocampus. Specific staining of bloodvessels and nerve-like cells were observed in the hippocampus of themonkeys administered with humanized anti-hTfR antibody No.3, humanizedanti-hTfR antibody No.3-2, humanized anti-hTfR antibody No.3(IgG4), andhumanized anti-hTfR antibody No.3-2 (IgG4) (FIG. 6b -e, respectively).Besides, no staining was observed in the hippocampus of the monkeyadministered with Herceptin as a control, indicating that the tissuestaining observed in FIG. 6b-e was specific for the humanized anti-hTfRantibodies (FIG. 6a ).

FIG. 7 shows the result of immunohistochemical staining of the humanizedanti-hTfR antibodies in the cerebellum. Specific staining of bloodvessels and Purkinje cells were observed in the cerebellum of themonkeys administered with humanized anti-hTfR antibody No.3, humanizedanti-hTfR antibody No.3-2, humanized anti-hTfR antibody No.3(IgG4), andhumanized anti-hTfR antibody No.3-2 (IgG4) (FIG. 7b -e, respectively).Besides, no staining was observed in the cerebellum of the monkeyadministered with Herceptin as a control, indicating that the tissuestaining observed in FIG. 7b-e was specific for the humanized anti-hTfRantibodies (FIG. 7a ).

FIG. 8 shows the result of immunohistochemical staining of the humanizedanti-hTfR antibodies in the medulla oblongata. Specific staining ofblood vessels and nerve-like cells were observed in the medullaoblongata of the monkeys administered with humanized anti-hTfR antibodyNo.3, humanized anti-hTfR antibody No.3-2, humanized anti-hTfR antibodyNo.3(IgG4), and humanized anti-hTfR antibody No.3-2 (IgG4) (FIG. 8b -e,respectively). Besides, no staining was observed in the medullaoblongata of the monkey administered with Herceptin as a control,indicating that the tissue staining observed in FIG. 8b-e was specificfor the humanized anti-hTfR antibodies (FIG. 8a ).

From the result of immunohistochemical staining of the cerebrum andcerebellum in Example 8 above, it was shown that anti-hTfR antibody No.3, pre-humanized mouse antibodies, can bind to hTfR occurring on theendothelium of blood vessel in the brain, and after binding to hTfR,pass through the blood-brain barrier into the brain parenchyma, andfurther be taken up into the brain parenchyma and nerve-like cell in thehippocampus, and into Purkinje cells in the cerebellum.

From the result of immunohistochemical staining in the cerebrum,hippocampus, cerebellum, and medulla oblongata in Example 15, it wasrevealed that the tested four humanized anti-hTfR antibodies obtained byhumanizing anti-hTfR antibody No.3 subjected to the experiment can bindto hTfR occurring on the endothelium of blood vessels of the brain, andafter binding to hTfR, pass through the blood-brain barrier and transferinto the brain parenchyma, and further, be taken up into nerve-likecells in the cerebral cortex; into the brain parenchyma and thenerve-like cells in the hippocampus; into Purkinje cells in thecerebellum; and into nerve-like cells in the medulla oblongata.

Example 16 Introduction of Mutation to Heavy Chain of HumanizedAnti-hTfR Antibody No. 3

In the heavy chain variable region of humanized anti-hTfR antibody No. 3set forth as SEQ ID NO: 37, when CDR1 was the one set forth as SEQ IDNO: 12, one amino acid in the amino acid sequence of the CDR1 wasreplaced with another amino acid while one amino acid in the amino acidsequence of framework region 3 was replaced with another amino acid toproduce a heavy chain of the humanized antibody. The heavy chain of thisnew antibody comprises the amino acid sequence set forth as SEQ ID NO:62 or 63 in CDR1 and comprises the amino acid sequence set forth as SEQID NO: 64 in framework region 3, as a result of replacing the two aminoacids constituting the amino acid sequence of the heavy chain ofhumanized anti-hTfR antibody No. 3 set forth in SEQ ID NO: 37. The heavychain of this new antibody (the heavy chain of humanized anti-hTfRantibody No. 3N) comprises the amino acid sequence set forth as SEQ IDNO: 66, and the variable region in the amino acid sequence comprises theamino acid sequence set forth as SEQ ID NO: 65.

Table 10 shows alignment between CDR1 set forth as SEQ ID NO: 12 in theheavy chain of humanized anti-hTfR antibody No. 3 and CDR1 set forth asSEQ ID NO: 62 in the heavy chain of humanized anti-hTfR antibody No. 3N.In the alignment, the amino acid threonine at position 5 from theN-terminal side was replaced with methionine.

Table 11 shows alignment between the framework region 3 (SEQ ID NO: 73)in the heavy chain of humanized anti-hTfR antibody No. 3 and theframework region 3 (SEQ ID NO: 64) in the heavy chain of humanizedanti-hTfR antibody No. 3N. In the alignment, the amino acid tryptophanat position 17 from the N-terminal side was replaced with leucine.

TABLE 10 Alignment of amino acid sequence of CDR1 CDR1Humanized anti-hTfR antibody No. 3 GYSFTNYW **** ***Humanized anti-hTfR antibody No. 3N GYSFMNYW * represents identicalamino acids.

TABLE 11  Alignment of amino acid sequence of framework region FR3Humanized anti-hTfR antibody No. 3 QVTISADKSISTAYLQWSSLKASDTAMYYC**************** ************* Humanized anti-hTfR antibody No. 3N QVTISADKSISTAYLQLSSLKASDTAMYYC * represents identical amino acids.

Example 17 Construction of Cells for Expression of Humanized Anti-hTfRAntibody No. 3N

A DNA fragment (SEQ ID NO: 67) was artificially synthesized, whichincluded a gene encoding the antibody heavy chain consisting of theamino acid sequence set forth as SEQ ID NO: 66. This DNA fragment wasadded, on its 5′ side, a MluI sequence and a sequence encoding a leaderpeptide in this order from the 5′ end, and a NotI sequence on its 3′side. The DNA thus synthesized was inserted into the vector pE-neo bythe method described in Example 11, and the vector thus obtained wasused as an expression vector for the heavy chain of humanized anti-hTfRantibody No. 3N, pE-neo(HC3)N. CHO cells were transformed by the methoddescribed in Example 12 using this pE-neo(HC3)N and the vector for lightchain expression, pE-hygr(LC3), constructed in Example 11, to obtain ahumanized anti-hTfR antibody No. 3N expressing cell line.

Example 18 Purification of Humanized Anti-hTfR Antibody No. 3N

A purified product of humanized anti-hTfR antibody No. 3N was obtainedby the method described in Example 13 using the humanized anti-hTfRantibody No. 3N expressing cell line obtained in Example 17. Besides,humanized anti-hTfR antibody No. 3N has the substitution of two aminoacids shown in Table 10 and Table 11 in the amino acid sequence of theheavy chain of humanized anti-hTfR antibody No. 3. On the other hand,the light chains of humanized anti-hTfR antibody No. 3N and humanizedanti-hTfR antibody No. 3 have identical amino acid sequences.

Example 19 Comparison of Affinity to Human TfR and Monkey TfR BetweenHumanized Anti-hTfR Antibody No. 3 and Humanized Anti-hTfR Antibody No.3N

The affinity of humanized anti-hTfR antibody No. 3 obtained in Example13 and humanized anti-hTfR antibody No. 3N obtained in Example 17 tohuman TfR and human TfR was measured by the method described in Example7. Table 12 shows the results of measurement of association rateconstant (kon), dissociation rate constant (koff) of each antibody, anddissociation constant (k_(D)) to human TfR. Besides, the measurementindicating the affinity of humanized anti-hTfR antibody No. 3 to humanTfR differed from that shown in Table 6, but is an experimental error.

TABLE 12 Affinity of humanized anti-hTfR antibody for human TfR kon(M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) Humanized anti-hTfR 8.47 × 10⁵ <1.0 × 10⁻⁷<1.0 × 10⁻¹² antibody 3 Humanized anti-hTfR 7.84 × 10⁵ <1.0 × 10⁻⁷ <1.0× 10⁻¹² antibody 3N

The affinity of humanized anti-hTfR antibody No. 3 obtained in Example13 and humanized anti-hTfR antibody No. 3N obtained in Example 17 tohuman TfR and monkey TfR was measured by the method described in Example7. Table 13 shows the results of measurement of association rateconstant (kon), dissociation rate constant (koff) of each antibody, anddissociation constant (k_(D)) to monkey TfR. Besides, the measurementindicating the affinity of humanized anti-hTfR antibody No. 3 to monkeyTfR differed from that shown in Table 7, but is an experimental error.

TABLE 13 Affinity of humanized anti-hTfR antibody for monkey TfR kon(M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) Humanized anti-hTfR 6.09 × 10⁵ 8.06 × 10⁻⁴1.32 × 10⁻⁹  antibody 3 Humanized anti-hTfR 3.86 × 10⁵ 1.96 × 10⁻⁵ 5.07× 10⁻¹¹ antibody 3N

When K_(D) with monkey TfR was compared between humanized anti-hTfRantibody No. 3N and humanized anti-hTfR antibody No. 3, the former was5.07×10⁻¹¹ M and the latter was 1.32×10⁻⁹ M; thus K_(D) of humanizedanti-hTfR antibody No. 3N with monkey TfR was approximately 1/30 of thatof humanized anti-hTfR antibody No. 3. On the other hand, when Kd withhuman TfR was compared between humanized anti-hTfR antibody No. 3N andhumanized anti-hTfR antibody No. 3, both were less than 1×10⁻¹² M; thusboth the antibodies had a high affinity to human TfR. Also, both theantibodies have a higher affinity to human TfR than the affinity tomonkey TfR.

As part of nonclinical trials to be conducted to develop humanizedanti-hTfR antibodies as pharmaceutical agents, pharmacological testsusing monkeys are often carried out. Because results of thesepharmacological tests using monkeys are used for judging the validity ofclinical trials using humans, it is preferable that behavior in thebodies of monkeys should be closer to behavior in the bodies of humans.As shown in the above results, humanized anti-hTfR antibody No. 3Nexhibits approximately 30 times higher affinity to monkey TfR than thatof humanized anti-hTfR antibody No. 3, and the behavior of humanizedanti-hTfR antibody No. 3N in the bodies of monkeys can therefore beregarded as being closer to its behavior in the bodies of humans. Thus,by using humanized anti-hTfR antibody No. 3N, results more reflectingbehavior in the bodies of humans are obtained in tests using monkeys.Thus, more beneficial results for making a decision to conduct clinicaltrials using humans are obtained by the tests using monkeys.

Example 20 Comparison of Transfer to Brain Tissue in Monkey BetweenHumanized Anti-hTfR Antibody No. 3 and Humanized Anti-hTfR Antibody No.3N

Each of humanized anti-hTfR antibody No. 3 obtained in Example 13 andhumanized anti-hTfR antibody No. 3N obtained in Example 17 wasintravenously administered once to a male crab-eating monkey at a dosageof 5.0 mg/kg, and 8 hours after the administration, whole bodyirrigation was carried out. After the irrigation, the brain and spinetissues including the medulla oblongata were excised. The brain tissuesthus excised were separated into the cerebral cortex, the cerebellum,the hippocampus and the medulla oblongata.

Measurement of the concentration of humanized anti-hTfR antibodiescontained in each tissue was carried out in accordance with themeasurement method described in Example 15.

The result of the concentration measurement of the anti-hTfR antibodiesin brain tissues is shown in Table 14. When the concentration in thecerebrum, the cerebellum, and the hippocampus was compared betweenhumanized anti-hTfR antibody No. 3N and humanized anti-hTfR antibody No.3, the concentration of humanized anti-hTfR antibody No. 3N wasapproximately 1.42 times in the cerebrum, approximately 1.56 times inthe cerebellum, and approximately 1.29 times in the hippocampus, higherthan that of humanized anti-hTfR antibody No. 3. In the medullaoblongata, their concentrations were almost equal. In the cervical spineas well, the concentration of humanized anti-hTfR antibody No. 3N wasapproximately 1.47 times higher than that of humanized anti-hTfRantibody No. 3. These results demonstrate that humanized anti-hTfRantibody No. 3N, compared with humanized anti-hTfR antibody No. 3, had aproperty to more efficiently pass through the blood brain barrier andaccumulate in the brain tissues of the cerebrum, the cerebellum, thehippocampus, and the like. Namely, the results show that by bindingthese antibodies to a pharmaceutical agent which needs to be broughtinto function in the brain tissues, humanized anti-hTfR antibody No. 3Nhad a property to let those pharmaceutical agents efficiently accumulatein the brain tissues.

TABLE 14 Concentration of anti-hTfR antibody in brain tissue (μg/g wetweight) Antibody Hippo- Medulla Cervical No. Cerebrum Cerebellum campusoblongata cord 3 0.670 0.610 0.490 0.589 0.589 3N 0.953 0.950 0.6310.586 0.672

Example 20-2 Preparation of Cells for Expression of hFc-HumanizedAnti-hTfR Antibody

A DNA fragment (SEQ ID NO: 72) was artificially synthesized, whichincluded a gene encoding the amino acid sequence of the human IgG Fcregion-added humanized anti-hTfR antibody 3N Fab heavy chain consistingof the amino acid sequence set forth as SEQ ID NO: 71. This DNA fragmentwas added, on its 5′ side, a MluI sequence and a sequence encoding aleader peptide in this order from the 5′ end, and a NotI sequence on its3′ side. The DNA thus synthesized was inserted into the vector pE-neo bythe method described in Example 11, and the vector thus obtained wasdesignated as pE-neo(Fc-Fab HC(3N)). CHO cells were transformed by themethod described in Example 12 using this pE-neo(Fc-Fab HC(3N)) and thevector for light chain expression, pE-hygr(LC3), constructed in Example11, to obtain a Fc-Fab(3N) expressing cell line.

Example 20-3 Preparation of hFc-Humanized Anti-hTfR Antibody

A purified product of Fc-Fab(3N) was obtained by the method described inExample 13 using the Fc-Fab(3N) expressing cell line.

Example 20-4 Comparison of Affinity of hFc-Humanized Anti-hTfR AntibodyBetween Human TfR and Monkey TfR

The affinity of the purified product of Fc-Fab(3N) obtained in Example20-3 to human TfR and monkey TfR was measured by the method described inExample 7. The results are shown in Table 14-2. Fc-Fab (3N) exhibited ahigh affinity (K_(D)<1.0×10⁻¹²) to human TfR. These results indicatethat Fc-Fab (3N) has a property that enables pharmaceutical agents whichneed to be brought into function in brain tissues to efficientlyaccumulate in the brain tissues, by binding such pharmaceutical agentsto one of these antibodies.

[Table 14-2]

TABLE 14-2 Affinity of hFc-humanized anti-hTfR antibody for human TfRand monkey TfR kon (M⁻¹s⁻¹) koff (s⁻¹) K_(D) (M) Human TfR 6.67 × 10⁵<1.0 × 10⁻⁷ <1.0 × 10⁻¹² Monkey TfR 2.43 × 10⁵ 2.09 × 10⁻⁴ 8.61 × 10⁻¹⁰

Example 20-5 Measurement of Transfer of hFc-Humanized Anti-hTfR Antibodyinto Brain Tissues in Monkey

The Fc-Fab (3N) obtained in Example 20-3 was intravenously administeredonce to a male crab-eating monkey at a dosage of 5.0 mg/kg, and 8 hoursafter the administration, whole body irrigation was carried out. Afterthe irrigation, the brain and spine tissues including the medullaoblongata were excised. The brain tissues thus excised were separatedinto the cerebral cortex, the cerebellum, the hippocampus and themedulla oblongata.

Measurement of the concentration of humanized anti-hTfR antibodiescontained in each tissue was carried out in accordance with themeasurement method described in Example 15.

The result of the concentration measurement of the anti-hTfR antibodiesin brain tissues is shown in Table 14-3. The concentrations of Fc-Fab(3N) in the cerebrum, the cerebellum, and the hippocampus were 0.80 μg/gin terms of a wet weight, 0.80 μg/g in terms of a wet weight and 1.05μg/g, respectively. The concentrations in the medulla oblongata and thespinal cord were 0.68 μg/g in terms of a wet weight and 0.67 μg/g interms of a wet weight, respectively. These results indicate that theanti-hTfR antibody can pass through BBB even when the humanizedanti-hTfR antibody which is Fab is bound, on the N-terminal sidethereof, to the Fc region. When a conjugate in which a desired substance(protein or physiologically active substance) is bound to Fab isintravitally administered and is instable, Fab is bound, on theN-terminal side thereof, to the Fc region, whereby it is possible tostabilize such a conjugate in the body, e.g. increase the bloodhalf-life of the conjugate while maintaining the property of passingthrough BBB.

TABLE 14-3 Concentration of hFc-humanized anti-hTfR antibody in braintissues (μg/g wet weight) Hippo- Medulla Cervical Cerebrum Cerebellumcampus oblongata cord hFc-humanized 0.80 0.80 1.05 0.67 0.68 anti-hTfRantibody

Example 21 Preparation of hI2S-Humanized Anti-hTfR Antibody FusionProtein Expression Cells

A DNA fragment was artificially synthesized having the nucleotidesequence set forth as SEQ ID NO:52, which included a gene encoding aprotein in which the humanized anti-hTfR antibody No. 3 heavy chainhaving the amino acid sequence set forth as SEQ ID NO:51 was linked, onthe C-terminal side thereof and via a linker sequence (Gly Ser), to hI2Shaving the amino acid sequence set forth as SEQ ID NO:50. This DNAfragment encoded a protein in which humanized anti-hTfR antibody heavychain having the amino acid sequence set forth as SEQ ID NO:247 waslinked, via a linker sequence (Gly Ser), to hI2S. This DNA fragment had,on its 5′ side, a MluI sequence, and a sequence encoding a leaderpeptide acting as a secretion signal in this order from the 5′ end, anda NotI sequence on its 3′ side. The DNA fragment was digested with MluIand NotI, and inserted into the vector pE-neo, between the MluI and NotIthereof, to construct pE-neo(HC-I2S-1).

A DNA fragment was artificially synthesized having the nucleotidesequence set forth as SEQ ID NO:54, which included a gene encoding aprotein in which the heavy chain of the humanized anti-hTfR antibody No.3N having the amino acid sequence set forth as SEQ ID NO:66 was linked,on the C-terminal side thereof and via a linker sequence (Gly Ser), tohI2S having the amino acid sequence set forth as SEQ ID NO:50. This DNAfragment encoded a protein in which humanized anti-hTfR antibody heavychain having the amino acid sequence set forth as SEQ ID NO:53 waslinked, via a linker sequence (Gly Ser), to hI2S. This DNA fragment had,on its 5′ side, a MluI sequence and a sequence encoding a leader peptideacting as a secretion signal in this order from the 5′ end, and a NotIsequence on its 3′ side. The DNA fragment was digested with MluI andNotI, and inserted into the vector pE-neo, between the MluI and NotIthereof, to construct pE-neo(HC-I2S-2).

CHO cells (CHO-K1: purchased from American Type Culture Collection) weretransformed with pE-neo(HC-I2S-3) and pE-hygr(LC3) which had beenprepared in Example 11, to obtain a cell line expressing a fusionprotein between hI2S and a humanized anti-hTfR antibody. This cell linewas designated hI2S-anti-hTfR antibody expressing cell line 3. Thefusion protein between hI2S and a humanized anti-hTfR antibody expressedby the cell lines was designated I2S-anti-hTfR antibody 3.

CHO cells were transformed with pE-neo(HC-I2S-3N) and pE-hygr(LC3) toobtain a cell line expressing a fusion protein between hI2S and ahumanized anti-hTfR antibody. This cell line was designated ashI2S-anti-hTfR antibody expressing cell line 3N. The fusion proteinbetween hI2S and a humanized anti-hTfR antibody expressed by the cellswas designated as I2S-anti-hTfR antibody 3N.

Example 22 Production of I2S-Anti-hTfR Antibodies

I2S-anti-hTfR antibodies were produced by the following method. With CDOptiCHO™ medium, hI2S-anti-hTfR antibody expressing cell lines 3 and 3Nobtained in Example 21 were diluted to the density of approximately2×10⁵ cells/mL, respectively. The cell suspensions, 200 mL, were addedto corresponding 1 L-conical flasks, and cultured for 6 to 7 days in awet environment of 37° C., 5% CO₂, 95% air with stirring at a rate ofabout 70 rpm. Each culture supernatant was collected by centrifugation,and filtered through a 0.22 μm filter (Millipore Inc.) to prepare theculture supernatant. To each culture supernatant thus obtained was addedfive column volumes of 20 mM Tris buffer (pH 8.0) containing 150 mLNaCl, and loaded onto a Protein A column (column volume: 1 mL, Bio-RadInc.) which had been equilibrated in advance with three column volumesof 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl. Then, the columnwas washed with five column volumes of the same buffer, and the adsorbedI2S-anti-hTfR antibody was eluted with four column volumes of 50 mMglycine buffer (pH 2.8) containing 150 mM NaCl. The pH of the eluatecontaining I2S-anti-hTfR antibody was adjusted to pH 7.0 with 1 M Trisbuffer (pH 8.0), and then the buffer was replaced with PBS buffer usingAmicon Ultra 30 kDa membrane (Millipore Inc.) to obtain purifiedproducts of I2S-anti-hTfR antibody 3 and I2S-anti-hTfR antibody 3N.

Example 23 Measurement of Affinity of I2S-Anti-hTfR Antibody to HumanTfR and Monkey TfR

The affinity of the I2S-anti-hTfR antibodies obtained in Example 22 tohuman TfR and monkey TfR was measured in accordance with the methoddescribed in Example 7. Table 15 shows the results of measurement ofassociation rate constant (kon), dissociation rate constant (koff) ofI2S-anti-hTfR antibody 3 and I2S-anti-hTfR antibody 3N, and dissociationconstant (k_(D)) to human TfR. Table 16 shows the results of measurementof association rate constant (kon), dissociation rate constant (koff) ofI2S-anti-hTfR antibody 3 and I2S-anti-hTfR antibody 3N, and dissociationconstant (k_(D)) to monkey TfR.

I2S-anti-hTfR antibody 3N exhibited approximately 5 times the affinityof I2S-anti-hTfR antibody to human TfR (Table 15). Further,I2S-anti-hTfR antibody 3N exhibited a much higher affinity to monkey TfRthan that of I2S-anti-hTfR antibody (Table 16). I2S-anti-hTfR antibody3N exhibited a higher affinity to human TfR and monkey TfR, than that ofI2S-anti-hTfR antibody 3.

TABLE 15 Affinity of I2S-anti-hTfR antibody to human TfR kon (M⁻¹s⁻¹)koff (s⁻¹) K_(D) (M) I2S-anti-hTfR antibody 3 8.29 × 10⁵ 1.04 × 10⁻⁴1.26 × 10⁻¹⁰ I2S-anti-hTfR antibody 3N 6.92 × 10⁵ 1.90 × 10⁻⁵ 2.75 ×10⁻¹¹

TABLE 16 Affinity of I2S-anti-hTfR antibody to monkey TfR kon (M⁻¹s⁻¹)koff (s⁻¹) K_(D) (M) I2S-anti-hTfR antibody 3 3.77 × 10⁵  4.10 × 10⁻⁴1.09 ×× 10⁻⁹ I2S-anti-hTfR antibody 3N 3.83 × 10⁵ <1.00 × 10⁻⁷ <1.00 ×10⁻¹²

Example 24 Comparison of Transfer to Brain Tissue in Monkey BetweenI2S-Anti-hTfR Antibody 3 and I2S-Anti-hTfR Antibody 3N

Each of I2S-anti-hTfR antibody 3 and I2S-anti-hTfR antibody 3N obtainedin Example 22 was intravenously administered once to a male crab-eatingmonkey at a dosage of 5.0 mg/kg, and 8 hours after the administration,whole body irrigation was carried out. After the irrigation, the brainand spine tissues including the medulla oblongata were excised. Thebrain tissues thus excised were separated into the cerebral cortex, thecerebellum, the hippocampus and the medulla oblongata.

Measurement of the concentration of I2S-anti-hTfR antibodies containedin each tissue was carried out largely following the procedure describedin Example 20. The result of the concentration measurement of theI2S-anti-hTfR antibodies in brain tissues is shown in Table 17. When theconcentration in the cerebrum, the cerebellum, the hippocampus, themedulla oblongata, and the cervical spine was compared betweenI2S-anti-hTfR antibody 3N and I2S-anti-hTfR antibody 3, theconcentration of I2S-anti-hTfR antibody 3N was approximately 2.12 timesin the cerebrum, approximately 1.97 times in the cerebellum,approximately 2.41 times in the hippocampus, 1.94 times in the medullaoblongata, and 1.63 times in the cervical spine, higher than that ofI2S-anti-hTfR antibody 3. This result demonstrates that I2S-anti-hTfRantibody 3N can pass through the blood-brain barrier more efficientlythan I2S-anti-hTfR antibody 3.

TABLE 17 Concentration of I2S-anti-hTfR antibody in brain tissue (μg/gwet weight) Hippo- Medulla Cervical Cerebrum Cerebellum campus oblongatacord I2S-anti-hTfR 0.224 0.249 0.183 0.229 0.188 antibody 3I2S-anti-hTfR 0.475 0.491 0.441 0.450 0.306 antibody 3N

Example 25 Preparation of Cells for Expression of hGAA-HumanizedAnti-hTfR Antibody Fusion Protein

A DNA fragment was artificially synthesized having the nucleotidesequence set forth as SEQ ID NO:59, which included a gene encoding aprotein in which the heavy chain of the humanized anti-hTfR antibody 3Nhaving the amino acid sequence set forth as SEQ ID NO:68 was linked, onthe C-terminal side thereof and via a linker sequence (Gly Ser), to hGAAhaving the amino acid sequence set forth as SEQ ID NO:55. This DNAfragment encoded a protein in which the heavy chain (IgG4) of thehumanized anti-hTfR antibody 3N having the amino acid sequence set forthas SEQ ID NO:58 was linked, via a linker sequence (Gly Ser), to hGAA.This DNA fragment had, on its 5′ side, a MluI sequence and a sequenceencoding a leader peptide acting as a secretion signal in this orderfrom the 5′ end, and a NotI sequence on its 3′ side. The DNA fragmentwas digested with MluI and NotI, and inserted into the vector pE-neo,between the MluI and NotI thereof, to construct pE-neo(HC-GAA-3N(IgG4)).

CHO cells (CHO-K1: purchased from American Type Culture Collection) weretransformed with pE-neo(HC-GAA-3N(IgG4)) and pE-hygr(LC3) constructed inExample 11 to obtain a cell line expressing a fusion protein betweenhGAA and a humanized anti-hTfR antibody. This cell line was designatedas hGAA-anti-hTfR antibody expressing cell line 3N(IgG4). The fusionprotein between hGAA and a humanized anti-hTfR antibody expressed by thecells was designated as GAA-anti-hTfR antibody 3N(IgG4).

Example 26 Production of GAA-Anti-hTfR Antibody 3N(IgG4)

GAA-anti-hTfR antibody 3N(IgG4) was produced largely following themethod described in Example 22 using the hGAA-anti-hTfR antibodyexpressing cell line 3N(IgG4) obtained in Example 25.

Example 27 Measurement of Affinity of hGAA-Anti-hTfR Antibody 3N (IgG4)to Human TfR and Monkey TfR

The affinity of the hGAA-anti-hTfR antibody 3N (IgG4) obtained inExample 26 to human TfR and monkey TfR was measured in accordance withthe method described in Example 23. The results of measurement of theaffinity of Fab GS-GAA prepared in Example 31 below to human TfR andmonkey TfR are also described here.

The hGAA-anti-hTfR antibody 3N (IgG4) exhibited a very high affinity toboth human TfR and monkey TfR. The results are shown in Table 17-2. FabGS-GAA also exhibited a high affinity to both human TfR and monkey TfR,but the affinity was lower than that of the hGAA-anti-hTfR antibody 3N(IgG4).

TABLE 17-2 Affinity of hGAA-anti-hTfR antibody 3N (IgG4) and Fab GS-GAAto human TfR and monkey TfR kon (1/Ms) koff (1/s) K_(D) (M) hGAA-anti-Human 4.86 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² hTfR TfR antibody Monkey2.03 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² 3N (IgG4) TfR Fab GS-GAA Human4.14 × 10⁵  1.67 × 10⁻⁴  4.04 × 10⁻¹⁰ TfR Monkey 3.29 × 10⁵  5.01 × 10⁻³ 1.52 × 10⁻⁸ TfR

Example 28 Assessment of Transfer of hGAA-Anti-hTfR Antibody 3N (IgG4)into Brain Tissues in Monkey

Transfer of the hGAA-anti-hTfR antibody 3N (IgG4) obtained in Example 26into brain tissues can be assessed in accordance with the methoddescribed in Example 24. Specifically, the hGAA-anti-hTfR antibody 3N(IgG4) was intravenously administered once to a male crab-eating monkeyat dosages of 5.0 mg/kg and 20 mg/kg. As a control agent, hGAAcommercially available as a therapeutic agent for Pompe's disease wasintravenously administered once to a male crab-eating monkey at dosagesof 5.0 mg/kg and 20 mg/kg. Three monkeys were administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 20 mg/kg, and onemonkey was administered with the other antibody. The monkeysadministered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of20 mg/kg were each subjected to whole body irrigation with aphysiological saline solution (Otsuka Pharmaceutical Co., Ltd.) 4 hours,8 hours and 24 hours after the administration. The other monkey wassubjected to whole body irrigation 8 hours after the administration.

After the irrigation, brain tissues including the medulla oblongata,spinal cord tissues and skeletal muscle tissues were excised. The braintissues were divided into the cerebral cortex, the cerebellum, thehippocampus, the medulla oblongata and the pontine. For the skeletonmuscle tissues, the rectus femoris muscle, the extensor digitorum longusmuscle, the soleus muscle, the triceps brachii muscle, the major psoasmuscle, the diaphragm and the lingual muscle were excised. One malecrab-eating monkey which had not been administered with a pharmaceuticalagent was subjected to whole body irrigation with a physiological salinesolution (Otsuka Pharmaceutical Co., Ltd.), and the cerebral cortex andthe rectus femoris muscle were excised. The excised tissues werehomogenized with T-PER (Thermo Fisher Scientific Inc.) containingProtease Inhibitor Cocktail (Sigma Inc.), and centrifuged to collect thesupernatant. Using the prepared supernatant, the concentrations of thehGAA-anti-hTfR antibody 3N (IgG4) and hGAA were measured by thefollowing ECL method, and immunohistochemical staining was performed.

Measurement of Concentration of Protein by ECL Method:

A rabbit anti-hGAA polyclonal antibody was SULFO-labeled with MSD GOLDSulfo-TAG NHS Ester (Meso Scale Discovery Inc.) to prepare aSULFO-labeled anti-hGAA antibody. Further, the rabbit anti-hGAApolyclonal antibody was Biotin-labeled with EZ-Link NHS-PEG4-Biotin(Thermo Fisher Scientific Inc.) to prepare a Biotin-labeled anti-hGAAantibody. A solution of a mixture of equal amounts of the SULFO-labeledanti-hGAA antibody and the Biotin-labeled anti-hGAA antibody wasprepared, the supernatant obtained as described above was added to thissolution, and the resulting mixture was incubated at room temperaturefor 1 hour to prepare an antibody reaction sample. A 1% BSA/PBS solutionwas added to each well of Streptavidin Gold Plate 96well (Meso ScaleDiagnostics Inc.) which is a plate coated with streptavidin, and theplate was left standing for 1 hour to block the plate.

After the blocking, each well of the plate was washed with 200 μL ofPBS-T (Sigma Inc.), and the antibody reaction sample was then added toeach well, and incubated at room temperature for 1 hour. After theincubation, each well of the plate was washed with PBS-T, a mixed liquidof the equal amounts of 4× Read buffer T (Meso scale Diagnostics Inc.)and water for injection (Otsuka Pharmaceutical Co., Ltd.) was thenadded, and the amount of luminescence from each well was measured usingSector™ Imager 6000 (Meso Scale Diagnostics Inc.). The amount of theantibody contained per one gram brain (wet weight) was calculated byproducing a standard curve based on measurements of standard samplescontaining known concentrations of the hGAA-anti-hTfR antibody 3N (IgG4)or hGAA, and interpolating the measurement of each sample with referenceto the standard curve to determine the amounts of the hGAA-anti-hTfRantibody 3N (IgG4) and hGAA.

The results of measurement of the concentrations by an ELC method areshown in Table 18. First, the results for brain tissues will bediscussed. In the monkey administered with the hGAA-anti-hTfR antibody3N (IgG4), the concentrations of the hGAA-anti-hTfR antibody 3N (IgG4)administered at dosages of 5 mg/kg and 20 mg/kg increased in aconcentration-dependent manner to 0.740 μg/g in terms of a wet weightand 1.42 μg/g in terms of a wet weight, respectively, in the cerebralcortex 8 hours after the administration. The same results were obtainedfor the cerebellum, the hippocampus, the medulla oblongata and thepontine. On the other hand, in the monkey administered with hGAA, theconcentrations of hGAA administered at dosages of 5 mg/kg and 20 mg/kgshowed almost equal values of 0.414 μg/g in terms of a wet weight and0.435 μg/g in terms of a wet weight, respectively, in the cerebralcortex 8 hours after the administration. The same results were obtainedfor the cerebellum, the hippocampus, the medulla oblongata and thepontine. In view of the fact that the value of the concentration ofintrinsic GAA in the cerebral cortex having the determined amount ofhGAA in the monkey which had not been administered with thepharmaceutical agent was 0.425 μg/g in terms of a wet weight, it isapparent that little of intravenously administered hGAA is taken up inthe brain. On the other hand, it is apparent that the intravenouslyadministered hGAA-anti-hTfR antibody 3N (IgG4) is taken up in the brainby passing through BBB.

Next, the results for skeletal muscle tissues will be discussed. In themonkey administered with each of the hGAA-anti-hTfR antibody 3N (IgG4)and hGAA at a dosage of 5 mg/kg, the concentration of the hGAA-anti-hTfRantibody 3N (IgG4) and hGAA in the rectus femoris muscle 8 hours afterthe administration was 0.311 μg/g in terms of a wet weight for thehGAA-anti-hTfR antibody 3N (IgG4) and 0.251 μg/g in terms of a wetweight for hGAA.

Namely, the concentration of the hGAA-anti-hTfR antibody 3N (IgG4) was1.23 times the concentration of hGAA. The same results were obtainedwhen these antibodies were administered at a dosage of 20 mg/kg.Further, the same results were obtained for the extensor digitorumlongus muscle, the soleus muscle, the triceps brachii muscle, the majorpsoas muscle, the diaphragm and the lingual muscle. Namely, it isapparent that the intravenously administered hGAA-anti-hTfR antibody 3N(IgG4) is taken up in skeletal muscle tissues in an amount larger thanthat of intravenously administered hGAA.

TABLE 18 Concentration of hGAA-anti-hTfR antibody 3N (IgG4) in braintissues and skeletal muscle (μg/g wet weight) Administered substancehGAA-anti-hTfR antibody 3N (IgG4) hGAA Dose 5 mg/kg 20 mg/kg 5 mg/kg 20mg/kg Elapsed time after administration (hr) Intrinsic 4 8 24 8 8 8 GAABrain Cerebral cortex 1.07 0.740 1.00 1.42 0.414 0.435 0.425 Cerebellum0.776 0.625 0.509 1.16 0.275 0.279 — Hippocampus 1.34 0.966 1.14 1.680.471 0.467 — Medulla oblongata 0.886 0.678 0.869 1.22 0.371 0.394 —Pontine 0.904 0.627 0.730 1.12 0.403 0.371 — Skeletal muscle Rectusfemoris muscle 0.445 0.311 0.458 0.425 0.251 0.377 0.140 Extensordigitorum 0.463 0.293 0.379 0.554 0.261 0.449 — longus muscle Soleusmuscle 0.527 0.361 0.377 0.485 0.277 0.320 — Triceps brachii muscle0.315 0.311 0.250 0.482 0.231 0.314 — Major psoas muscle 0.301 0.2410.275 0.383 0.238 0.225 — Diaphragm 0.289 0.212 0.238 0.339 0.199 0.381— Lingual muscle 0.882 0.631 0.794 1.10 0.316 0.915 —

Immunohistochemical Staining:

Immunohistochemical staining of the hGAA-anti-hTfR antibody 3N (IgG4)and hGAA in brain tissues was carried out following the proceduresdescribed below as a whole. The brain tissues obtained as describedabove were rapidly frozen to −80° C. in Tissue-Tek Cryo 3DM (SakuraFinetek Inc.), the frozen block thus obtained was sliced into 4-μmsections with LEICA CM 1860 UV Cryostat (Leica Biosystems NusslochInc.), and the tissue sections were then applied to MAS-coated glassslides (Matsunami Glass Inc.). The tissue sections were reacted with 4%paraformaldehyde (Wako Pure Chemical Industries Inc.) at 4° C. for 5minutes, and affixed onto glass slides. Subsequently, the tissuesections were reacted with a methanol solution containing 0.3% hydrogenperoxide (Wako Pure Chemical Industries Inc.) for 30 minutes toinactivate intrinsic peroxidase. The glass slides were then blocked byreacting SuperBlock blocking buffer in PBS at room temperature for 30minutes. The tissue sections were then reacted with a rabbit anti-hGAApolyclonal antibody as a primary antibody at 4° C. for 16 hours. Thetissue sections were then treated with CSAII Rabbit Link (Agilent Inc.)and a CSAII Biotin-free Tyramide Signal Amplification System kit(Agilent Inc.). The tissue sections were then reacted withAnti-Fluorescein HRP at room temperature for 15 minutes. The tissuesections were allowed to develop a color with a DAB substrate(3,3′-diaminobenzidine, Vector Laboratories Inc.), counterstained with aMayer's hematoxylin solution (Merck Inc.), dehydrated, cleared,embedded, and observed with a microscope. It is to be noted thatimmunohistochemical staining was carried out only on the cerebellum ofmonkeys administered with the hGAA-anti-hTfR antibody 3N (IgG4) at adosage of 20 mg/kg and administered with hGAA at a dosage of 20 mg/kg.Therefore, the elapsed time after administration is 8 hours for both theantibodies.

FIG. 9 shows the results of immunohistochemical staining of thehGAA-anti-hTfR antibody 3N (IgG4) and hGAA in the cerebellum. In thecerebellum of the monkey administered with the hGAA-anti-hTfR antibody3N (IgG4), specific staining of blood vessels and Purkinje cells wasobserved (FIG. 9a ). On the other hand, in the cerebellum of the monkeyadministered with hGAA, such staining was not observed (FIG. 9b ). Theseresults indicate that by fusing hGAA, which normally does not passthrough the blood-brain barrier, with the anti-hTfR antibody 3N (IgG4),the hGGA can be allowed to pass through the blood-brain barrier, andtaken up by Purkinje cells in the cerebellum.

Example 29 Assessment of Pharmacological Effect of hGAA-Anti-hTfRAntibody 3N (IgG4) in Mouse: Determination of Concentration of Glycogen(1)

Based on knockout mice as an animal model of Pompe's disease in which anacidic α-glucosidase (GAA) gene is disrupted, mice lacking a GAA gene inhomology and having a chimera TfR gene in heterology (GAA-KO/hTfR-KImice) were prepared by replacing a gene encoding the extracellularregion of mouse TfR with a gene encoding the extracellular region of thehuman TfR receptor gene by the method described in Example 7-2. The micewere divided into seven groups, and the hGAA-anti-hTfR antibody 3N(IgG4) or hGAA was intravenously administered thereto in accordance withthe usage and dosage shown in Table 19. Normal mice which had not beenadministered with a pharmaceutical agent were categorized as normalcontrols (first group), and GAA-KO/hTfR-KI mice which had not beenadministered with a pharmaceutical agent were categorized as diseasecontrols (second group). Each group included five mice. Further, maleand female mice which were 9 to 14-week old (at the start ofadministration) were used.

TABLE 19 Grouping of mice Interval of Number of Test Dosage adminis-adminis- Group substance Mouse (mg/kg) tration trations 1 — Normal mouse— — — 2 — GAA-KO/ — — — hTfR-KI mouse 3 hGAA GAA-KO/ 20 2 weeks 4 weekshTfR-KI mouse 4 hGAA-anti- GAA-KO/ 2.5 5 hTfR anti- hTfR-KI 5.0 6 body3N mouse 10 7 (IgG4) 20

At the second and third administration of the test substance,diphenhydramine was administered at a dosage of 10 mL/kg to the mouse 10to 20 minutes before the administration. This treatment is aimed atpreventing occurrence of an immunological reaction such as ananaphylactic shock in experimental animals due to administration of thetest substance. 13 days after the last administration, the mouse wasdecapitated under isoflurane anesthesia, and the right brain and thecervical part of spinal cord were quickly excised, and then quicklyimmersed in liquid nitrogen to be rapidly frozen. After the freezing,the wet weight of the tissues of each of the organs was measured. Forthe heart, the diaphragm, the liver, the spleen, the quadriceps femorismuscle, the soleus muscle, the rectus femoris muscle, the extensordigitorum longus muscle and the gastrocnemius muscle (collected skeletalmuscle: right leg), blood was sufficiently removed by exsanguination,tissues were then collected, and washed with a physiological salinesolution, and the wet weight of the tissues of each of the organs wasmeasured. After the measurement, the organs were each sealed in a 1.5 mLtube, and instantaneously frozen with liquid nitrogen.

Water for injection was added to the tissues of each organ in an amount20 times the wet weight of the tissues, and the tissues were homogenizedusing a bead grinder. The homogenized tissues were transferred into acentrifugal tube, heated at 100° C. for 5 minutes, and left standing onice. Subsequently, the tissues were centrifuged at 13000 rpm at 4° C.for 5 minutes, and the supernatant was collected. The supernatant wasfrozen and preserved at a temperature of −70° C. or lower.

The concentration of glycogen contained in the supernatant was measuredby Glycogen Assay Kit (Viovision Inc.). The results of the measurementare shown in FIGS. 10 and 11. FIG. 10 shows the concentrations ofglycogen in the right brain, the cervical part of spinal cord, theheart, the diaphragm, the liver and the spleen (FIGS. 10a to 10f ,respectively). Further, FIG. 11 shows the concentrations of glycogen inthe quadriceps femoris muscle, the soleus muscle, the rectus femorismuscle, the extensor digitorum longus muscle and the gastrocnemiusmuscle (FIGS. 11a to 11 e, respectively). However, the group of miceadministered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of20 mg/kg were excluded because they died 5 days after the lastadministration.

The results in FIGS. 10 and 11 will be discussed below.

Brain:

The concentrations of glycogen in the brain tissues of the normalcontrol group, the disease control group, the group of mice administeredwith hGAA at a dosage of 20 mg/kg, and the groups of mice administeredwith the hGAA-anti-hTfR antibody 3N (IgG4) at dosages of 2.5, 5.0, 10and 20 mg/kg were 0.0815 mg/g, 3.08 mg/g, 2.36 mg/g, 2.07 mg/g, 1.00mg/g, 0.891 mg/g and 0.592 mg/g, respectively, in terms of a wet weight(FIG. 10(a)). The groups of mice administered with the hGAA-anti-hTfRantibody 3N (IgG4) at a dosage of not lower than 5.0 mg/kg showed asignificant decrease in concentration of glycogen in the tissues ascompared to the disease control group and the group of mice administeredwith hGAA at a dosage of 20 mg/kg. Further, the decrease of the glycogenconcentration by the hGAA-anti-hTfR antibody 3N (IgG4) wasdosage-dependent.

Cervical Part of Spinal Cord:

The concentrations of glycogen in the cervical cord tissues of thenormal control group, the disease control group, the group of miceadministered with hGAA at a dosage of 20 mg/kg, and the groups of miceadministered with the hGAA-anti-HTFR antibody 3N (IgG4) at dosages of2.5, 5.0, 10 and 20 mg/kg were 0.0459 mg/g, 3.10 mg/g, 2.68 mg/g, 2.26mg/g, 1.77 mg/g, 1.06 mg/g and 0.795 mg/g, respectively, in terms of awet weight (FIG. 10(b)). All the groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) showed a significant decrease inconcentration of glycogen in the tissues as compared to the diseasecontrol group. Further, the groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of not lower than 5.0mg/kg showed a significant decrease in concentration of glycogen in thetissues as compared to the group of mice administered with hGAA at adosage of 20 mg/kg. Further, the decrease of the glycogen concentrationby the hGAA-anti-hTfR antibody 3N (IgG4) was dosage-dependent.

Heart:

The concentrations of glycogen in the heart of the normal control group,the disease control group, the group of mice administered with hGAA at adosage of 20 mg/kg, and the groups of mice administered with thehGAA-anti-HTFR antibody 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20mg/kg were 0.0816 mg/g, 32.4 mg/g, 4.54 mg/g, 12.3 mg/g, 3.45 mg/g, 1.10mg/g and 0.205 mg/g, respectively, in terms of a wet weight (FIG.10(c)). All the groups of mice administered with the hGAA-anti-hTfRantibody 3N (IgG4) showed a significant decrease in concentration ofglycogen in the tissues as compared to the disease control group. Thegroup of mice administered with the hGAA-anti-hTfR antibody 3N (IgG4) ata dosage of 20 mg/kg showed a significant decrease in concentration ofglycogen as compared to the group of mice administered with hGAA at adosage of 20 mg/kg.

Diaphragm:

The concentrations of glycogen in the diaphragm tissues of the normalcontrol group, the disease control group, the group of mice administeredwith hGAA at a dosage of 20 mg/kg, and the groups of mice administeredwith the hGAA-anti-hTfR antibody 3N (IgG4) at dosages of 2.5, 5.0, 10and 20 mg/kg were 0.0824 mg/g, 14.1 mg/g, 6.96 mg/g, 12.4 mg/g, 5.57mg/g, 2.68 mg/g and 0.564 mg/g, respectively, in terms of a wet weight(FIG. 10(d)). The groups of mice administered with the hGAA-anti-hTfRantibody 3N (IgG4) at a dosage of not lower than 5.0 mg/kg showed asignificant decrease in concentration of glycogen in the tissues ascompared to the disease control group. The group of mice administeredwith the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 10 mg/kgshowed a significant decrease in concentration of glycogen as comparedto the group of mice administered with hGAA at a dosage of 20 mg/kg.

Liver:

The concentrations of glycogen in the liver of the normal control group,the disease control group, the group of mice administered with hGAA at adosage of 20 mg/kg, and the groups of mice administered with thehGAA-anti-HTFR antibody 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20mg/kg were 1.16 mg/g, 22.5 mg/g, 2.65 mg/g, 8.88 mg/g, 2.95 mg/g, 3.01mg/g and 1.46 mg/g, respectively, in terms of a wet weight (FIG. 10(e)).All the groups of mice administered with the hGAA-anti-HTFR antibody 3N(IgG4), as well as the group of mice administered with hGAA at a dosageof 20 mg/kg, showed a significant decrease in concentration of glycogenin the tissues as compared to the disease control group.

Spleen:

The concentrations of glycogen in the spleen of the normal controlgroup, the disease control group, the group of mice administered withhGAA at a dosage of 20 mg/kg, and the groups of mice administered withthe hGAA-anti-HTFR antibody 3N (IgG4) at dosages of 2.5, 5.0, 10 and 20mg/kg were 0.0295 mg/g, 3.15 mg/g, 0.182 mg/g, 0.368 mg/g, 0.165 mg/g,0.122 mg/g and 0.0990 mg/g, respectively, in terms of a wet weight (FIG.10(f)). All the groups of mice administered with the hGAA-anti-HTFRantibody 3N (IgG4), as well as the group of mice administered with hGAAat a dosage of 20 mg/kg, showed a significant decrease in concentrationof glycogen in the tissues as compared to the disease control group.

Quadriceps Femoris Muscle:

The concentrations of glycogen in the quadriceps femoris muscle tissuesof the normal control group, the disease control group, the group ofmice administered with hGAA at a dosage of 20 mg/kg, and the groups ofmice administered with the hGAA-anti-HTFR antibody 3N (IgG4) at dosagesof 2.5, 5.0, 10 and 20 mg/kg were 0.0529 mg/g, 8.66 mg/g, 7.41 mg/g,6.40 mg/g, 2.68 mg/g, 1.71 mg/g and 0.282 mg/g, respectively, in termsof a wet weight (FIG. 11(a)). The groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of not lower than 5.0mg/kg showed a significant decrease in concentration of glycogen in thetissues as compared to the disease control group and the group of miceadministered with hGAA at a dosage of 20 mg/kg. The group of miceadministered with hGAA at a dosage of 20 mg/kg did not show asignificant decrease in concentration of glycogen in the tissues ascompared to the disease control group.

Gastrocnemius Muscle:

The concentrations of glycogen in the gastrocnemius muscle tissues ofthe normal control group, the disease control group, the group of miceadministered with hGAA at a dosage of 20 mg/kg, and the groups of miceadministered with the hGAA-anti-HTFR antibody 3N (IgG4) at dosages of2.5, 5.0, 10 and 20 mg/kg were 0.173 mg/g, 7.68 mg/g, 5.09 mg/g, 5.23mg/g, 2.40 mg/g, 1.58 mg/g and 0.221 mg/g, respectively, in terms of awet weight (FIG. 11(b)). The groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of not lower than 5.0mg/kg showed a significant decrease in concentration of glycogen in thetissues as compared to the disease control group. The group of miceadministered with hGAA at a dosage of 20 mg/kg did not show asignificant decrease in concentration of glycogen in the tissues ascompared to the disease control group.

Soleus Muscle:

The concentrations of glycogen in the soleus muscle tissues of thenormal control group, the disease control group, the group of miceadministered with hGAA at a dosage of 20 mg/kg, and the groups of miceadministered with the hGAA-anti-HTFR antibody 3N (IgG4) at dosages of2.5, 5.0, 10 and 20 mg/kg were 0.116 mg/g, 12.08 mg/g, 2.233 mg/g, 7.20mg/g, 2.74 mg/g, 1.07 mg/g and 0.378 mg/g, respectively, in terms of awet weight (FIG. 11(c)). All the groups of mice administered with thehGAA-anti-HTFR antibody 3N (IgG4) showed a significant decrease inconcentration of glycogen in the tissues as compared to the diseasecontrol group. Further, the groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of not lower than 5.0mg/kg, as well as the group of mice administered with hGAA at a dosageof 20 mg/kg, showed a significant decrease in concentration of glycogenin the tissues.

Rectus Femoris Muscle:

The concentrations of glycogen in the rectus femoris muscle tissues ofthe normal control group, the disease control group, the group of miceadministered with hGAA at a dosage of 20 mg/kg, and the groups of miceadministered with the hGAA-anti-HTFR antibody 3N (IgG4) at dosages of2.5, 5.0, 10 and 20 mg/kg were 0.0419 mg/g, 9.68 mg/g, 8.23 mg/g, 4.13mg/g, 2.56 mg/g, 0.895 mg/g and 0.277 mg/g, respectively, in terms of awet weight (FIG. 11(d)). All the groups of mice administered with thehGAA-anti-HTFR antibody 3N (IgG4) showed a significant decrease inconcentration of glycogen in the tissues as compared to the diseasecontrol group and the group of mice administered with hGAA at a dosageof 20 mg/kg. The group of mice administered with hGAA at a dosage of 20mg/kg did not show a significant decrease in concentration of glycogenin the tissues as compared to the disease control group.

Extensor Digitorum Longus Muscle:

The concentrations of glycogen in the extensor digitorum longus muscletissues of the normal control group, the disease control group, thegroup of mice administered with hGAA at a dosage of 20 mg/kg, and thegroups of mice administered with the hGAA-anti-HTFR antibody 3N (IgG4)at dosages of 2.5, 5.0, 10 and 20 mg/kg were 0.0950 mg/g, 10.4 mg/g,9.11 mg/g, 4.79 mg/g, 2.59 mg/g, 1.34 mg/g and 0.188 mg/g, respectively,in terms of a wet weight (FIG. 11(e)). All the groups of miceadministered with the hGAA-anti-HTFR antibody 3N (IgG4) showed asignificant decrease in concentration of glycogen in the tissues ascompared to the disease control group. Further, the groups of miceadministered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage ofnot lower than 5.0 mg/kg showed a significant decrease in concentrationof glycogen in the tissues as compared to the group of mice administeredwith hGAA at a dosage of 20 mg/kg. The group of mice administered withhGAA at a dosage of 20 mg/kg did not show a significant decrease inconcentration of glycogen in the tissues as compared to the diseasecontrol group.

Example 30 Assessment of Pharmacological Effect of hGAA-Anti-hTfRAntibody 3N (IgG4) in Mouse: Histopathological Assessment (2)

The left brain, the cervical part of spinal cord, the quadriceps femorismuscle, the soleus muscle and the extensor digitorum longus muscle,which had been collected in Example 29, was immersed in a sufficientamount of a 10% neutral formalin buffer solution. After about 24 hours,the 10% neutral formalin buffer solution was replaced. 3 days aftercollection of the tissues, the tissues were immersed in 80% ethanol, anda 48-hour delay timer was programmed, so that the ethanol was replacedwith toluene by an automatic fixing and embedding device (SakuraRotary), the tissues were finally immersed in paraffin, and the samplesthus obtained were subjected to PAS staining and HE staining.

The results of PAS staining will be discussed below.

Cerebral Cortex:

As for PAS stainability in cells, all of the disease control group andthe group of mice administered with hGAA at a dosage of 20 mg/kg hadmoderate stainability, whereas the groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 2.5 to 20 mg/kg showeda decrease in stainability. As for PAS stainability in blood vessels,the group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg showed a decrease in stainability.

Hippocampus/Dentate Gyrus and Basal Ganglia

The group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg showed a decrease in PAS stainability incells and blood vessels as compared to the disease control group. In thebasal ganglia, the group of mice administered with the hGAA-anti-hTfRantibody 3N (IgG4) at a dosage of 2.5 mg/kg and the group of miceadministered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of5.0 mg/kg also showed a decrease in stainability.

Interbrain (Thalamus/Hypothalamus):

The groups of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 2.5 to 20 mg/kg showed a decrease in PASstainability as compared to the disease control group.

Midbrain-Pontine:

The groups of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 5.0 to 20 mg/kg showed a decrease in PASstainability.

Medulla Oblongata:

The group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg showed a decrease in PAS stainability ascompared to the disease control group.

Cerebellum:

In the Purkinje cell layer/granulosa layer, the groups of miceadministered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of2.5 to 20 mg/kg tended to show a decrease in PAS stainability in cellson the periphery of Purkinje cells as compared to the disease controlgroup. In the white matter, the groups of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at dosages of 2.5 and 20 mg/kg tendedto show a decrease in PAS stainability.

Quadriceps Femoris Muscle:

The groups of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at dosages of 5.0 and 10 mg/kg tended to show a decrease in PASstainability as compared to the disease control group, and the group ofmice administered with the hGAA-anti-hTfR antibody 3N (IgG4) at a dosageof 20 mg/kg showed a remarkable decrease in stainability.

Soleus Muscle:

The group of mice administered with hGAA at a dosage of 20 mg/kg and thegroup of mice administered with the hGAA-anti-hTfR antibody 3N (IgG4) ata dosage of 10 mg/kg showed a decrease in PAS stainability as comparedto the disease control group, and the group of mice administered withthe hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 20 mg/kg showed aremarkable decrease in stainability.

Next, the results of HE staining will be discussed below.

Cerebral Cortex:

Cerebral cortex: the groups of mice administered with the hGAA-anti-hTfRantibody 3N (IgG4) at a dosage of 5.0 to 20 mg/kg tended to show areduction in growth of cell tumor as compared to the disease controlgroup. Cerebrum white matter (corpus callosum, fimbria hippocampi andanterior commissure) and brain ventricle: the group of mice administeredwith hGAA at a dosage of 20 mg/kg and the groups of mice administeredwith hGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 5.0 to 20 mg/kgshowed a decrease in vacuole content of the white matter as compared tothe disease control group.

Only the group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg tended to show a reduction in growth ofcell tumor as compared to the medulla oblongata disease control group.

Cerebellum:

The group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg tended to show a reduction in growth ofcell tumor in the white matter.

Quadriceps Femoris Muscle:

The group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 10 mg/kg tended to show a reduction invacuole/lacuna formation in the muscle fibers as compared to the diseasecontrol group, and the group of mice administered with thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of 20 mg/kg showed afurther reduction in vacuole/lacuna formation in the muscle fibers.

Soleus Muscle:

The group of mice administered with hGAA at a dosage of 20 mg/kg and thegroups of mice administered with the hGAA-anti-hTfR antibody 3N (IgG4)at a dosage of 5.0 to 10 mg/kg showed a reduction in vacuole/lacunaformation in the muscle fibers as compared to the disease control group,and the group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 20 mg/kg showed little reduction in vacuole/lacunaformation in the muscle fibers.

Extensor Digitorum Longus Muscle:

The group of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 5.0 mg/kg showed a reduction in vacuole/lacunaformation in the muscle fibers as compared to the disease control group,and the groups of mice administered with the hGAA-anti-hTfR antibody 3N(IgG4) at a dosage of 10 to 20 mg/kg showed a further reduction invacuole/lacuna formation in the muscle fibers.

The above results of the pharmacological effect of the hGAA-anti-hTfRantibody 3N (IgG4) in the mouse indicate that administration of thehGAA-anti-hTfR antibody 3N (IgG4) at a dosage of not lower than 5 mg/kgis more effective as a therapeutic agent for Pompe's disease from ahistopathological point of view than administration of commerciallyavailable hGAA at a dosage of 20 mg/kg, and exhibits an extremelyremarkable effect in the central nervous system and the skeletal muscletissues.

Example 31 Preparation of Fusion Protein Between Fab Anti-hTfR Antibody3N and hGAA (Fab GS-GAA)

A DNA fragment having a nucleotide sequence set forth as SEQ ID NO: 89and comprising a gene encoding a fusion protein between hGAA and the Fabheavy chain of a humanized anti-hTfR antibody was artificiallysynthesized, and inserted into the pE-neo vector between MIuI and NotIthereof to construct an expression vector pE-neo (Fab HC-GS-GAA). Here,into the gene encoding the fusion protein, a nucleotide sequenceencoding a leader peptide acting as a secretion signal was introduced onthe 5′-terminal side of the gene. CHO cells (CHO-K1: purchased fromAmerican Type Culture Collection) were transformed with the pE-neo (FabHC-GS-GAA) and the pE-hygr (LC3) constructed in Example 11 to obtain acell line expressing a fusion protein between hGAA and an anti-hTfRantibody 3N being Fab (Fab GS-GAA). A culture supernatant was obtainedby culturing the expression cell line in accordance with the methoddescribed in Example 22. The culture supernatant was loaded onCaptureSelect IgG-CH1 column being a CH1 affinity column (column volume:about 1.0 mL, Thermo Fisher Scientific Inc.), which had beenequilibrated with five column volumes of a 20 mM HEPES-NaOH buffersolution (pH: 7.0) containing 100 mM NaCl, to adsorb the fusion proteinto the CH1 affinity column. The column was then washed by supplying fivecolumn volumes of a 20 mM HEPES-NaOH buffer solution (pH: 7.0)containing 100 mM NaCl. The fusion protein adsorbed to the CH1 affinitycolumn was then eluted with five column volumes of a 100 mM glycinebuffer solution (pH: 3.0) containing 50 mM NaCl. The eluate wasimmediately neutralized by adding the eluate in a container which hadbeen supplied with a 1 M HEPES-NaOH buffer solution (pH: 7.5) inadvance. The product thus obtained was used as a purification product ofFab GS-GAA in the following experiments.

Example 32 Assessment of Pharmacological Effect of Fab GS-GAA in Mouse

To the mice (GAA-KO/hTfR-KI mice) described in Example 28, the FabGS-GAA obtained in Example 31, or hGAA was intravenously administered inaccordance with the usage and dosage described in Table 19-2. Normalmice which had not been administered with a pharmaceutical agent werecategorized as normal controls (first group), and GAA-KO/hTfR-KI micewhich had not been administered with a pharmaceutical agent werecategorized as disease controls (second group). Each group includedthree or four mice. Further, male and female mice which were 9 to14-week old (at the start of administration) were used. One week afterthe administration, the right brain, the heart, the diaphragm, thequadriceps femoris muscle, the soleus muscle and the rectus femorismuscle were collected by the method described in Example 29, theconcentration of glycogen contained in the tissues of each of the organswas measured. The results of the measurement are shown in FIG. 12. Ascompared to the group of mice administered with hGAA, the group of miceadministered with Fab GS-GAA showed a remarkable decrease inconcentration of glycogen in all the organs whose glycogen concentrationwas measured. The group of mice administered with Fab GS-GAA showed aparticularly remarkable decrease in concentration of glycogen in theright brain and the heart. Further, the group of mice administered withFab GS-GAA at a dosage of 5.0 mg/kg showed a remarkable decrease inconcentration of glycogen in the right brain and the heart as comparedto the group of mice administered with hGAA at a dosage of 20 mg/kg.These results indicate that Fab GS-GAA can be used as a therapeuticagent for Pompe's disease, particularly as a therapeutic agent forPompe's disease accompanied by brain and/or heart dysfunction.

TABLE 19-2 Grouping of mice Group Test substance Mouse Dosage (mg/kg) 1— Normal mouse — 2 — GAA-KO/hTfR-KI mouse — 3 hGAA GAA-KO/hTfR-KI mouse20 4 Fab GS-GAA GAA-KO/hTfR-KI mouse 5.0 5 20

Example 33 Preparation of Fusion Proteins Between Various HumanLysosomal Enzymes and Humanized Anti-hTfR Antibody

A DNA fragment was artificially synthesized which had a nucleotidesequence comprising a gene encoding fusion proteins between varioushuman lysosomal enzymes and the heavy chain of a humanized anti-hTfRantibody (note: the expression vector was a single-chain humanizedanti-hTfR antibody when the fusion protein was scFab-IDUA). These fusionproteins have amino acid sequences set forth as SEQ ID NONS shown inTables 20-A to 20-C, and are designated in the column of “fusion proteindesignation” in Tables 20-A to 20-C. The synthesized DNA fragment wasinserted into the pE-neo vector between MIuI and NotI thereof toconstruct an expression vector. Here, into the gene encoding the fusionprotein, a nucleotide sequence encoding a leader peptide acting as asecretion signal was introduced on the 5′-terminal side of the gene. Thedesignations of the expression vectors are shown in Tables 20-A to 20-C.CHO cells (CHO-K1: purchased from American Type Culture Collection) weretransformed with each expression vector obtained and the pE-hygr (LC3)constructed in Example 11 to obtain a cell line expressing fusionproteins between various lysosomal enzymes and a humanized anti-hTfRantibody. A culture supernatant was obtained by culturing the expressioncell line in accordance with the method described in Example 22.

The fusion proteins between the lysosomal enzyme and the humanizedanti-hTfR antibody, which were contained in the culture supernatant,were produced by the following purification method 1 for those havingFab as an antibody, and by the following purification method 2 for thosewith an antibody including the full length of the heavy chain.

Purification method 1: The culture supernatant was loaded onCaptureSelect IgG-CH1 column being a CH1 affinity column (column volume:about 1.0 mL, Thermo Fisher Scientific Inc.), which had beenequilibrated with five column volumes of a 20 mM HEPES-NaOH buffersolution (pH: 7.0) containing 100 mM NaCl, to adsorb the fusion proteinto the CH1 affinity column. The column was then washed by supplying fivecolumn volumes of a 20 mM HEPES-NaOH buffer solution (pH: 7.0)containing 100 mM NaCl. The fusion protein adsorbed to the CH1 affinitycolumn was then eluted with five column volumes of a 100 mM glycinebuffer solution (pH: 3.0) containing 50 mM NaCl. The eluate wasimmediately neutralized by adding the eluate in a container which hadbeen supplied with a 1 M HEPES-NaOH buffer solution (pH: 7.5) inadvance.

Purification method 2: The culture supernatant was loaded on MabSelectSuRe LX column being a protein A affinity column (column volume: about1.0 mL, GE Healthcare Inc.), which had been equilibrated with fivecolumn volumes of a 20 mM HEPES-NaOH buffer solution (pH: 7.0)containing 100 mM NaCl, to adsorb the fusion protein to the protein A.The column was then washed by supplying five column volumes of a 20 mMHEPES-NaOH buffer solution (pH: 7.0) containing 100 mM NaCl. The fusionprotein adsorbed to the protein A was then eluted with five columnvolumes of a 100 mM glycine buffer solution (pH: 3.0) containing 50 mMNaCl. The eluate was immediately neutralized by adding the eluate in acontainer which had been supplied with a 1 M HEPES-NaOH buffer solution(pH: 7.5) in advance.

Using the thus-obtained purification products of fusion proteins, thefollowing experiments were conducted. Further, the thus-obtained fusionproteins between lysosomal enzymes and a humanized anti-hTfR antibodywere designated as shown in the rightmost column in Tables 20-A to 20-C.

TABLE 20(A) List of fusion proteins between various human lysosomalenzymes and humanized anti-hTfR antibody Designation of lysosomalDesignation of Light-chain enzyme-humanized Designation of SEQDesignation of expression expression anti-hTfR antibody lysosome ID NOfusion protein vector vector fusion protein hGAA 89 Fab HC-GS3-GAApE-neo (Fab HC-GS3-GAA) pE-hygr (LC3) Fab GS3-GAA hIDUA 90 IgG4 HC-IDUApE-neo (IgG4 HC-IDUA) pE-hygr (LC3) IgG4 IDUA 91 IgG4 HC-GS8-IDUA pE-neo(IgG4 HC-GS8-IDUA) pE-hygr (LC3) IgG4 GS8-IDUA 92 IgG4 HC-GS20-IDUApE-neo (IgG4 HC-GS20-IDUA) pE-hygr (LC3) IgG4 GS20-IDUA 93 FabHC-GS3-IDUA pE-neo (Fab HC-GS3-IDUA) pE-hygr (LC3) Fab GS3-IDUA 94 FabHC-GS5-IDUA pE-neo (Fab HC-GS5-IDUA) pE-hygr (LC3) Fab GS5-IDUA 95 FabHC-GS10-IDUA pE-neo (Fab HC-GS10-IDUA) pE-hygr (LC3) Fab GS10-IDUA 96Fab HC-GS20-IDUA pE-neo (Fab HC-GS20-IDUA) pE-hygr (LC3) Fab GS20-IDUA97 Tandem Fab HC-IDUA pE-neo (Tandem Fab HC-IDUA) pE-hygr (LC3) TandemFab IDUA 99 scFab-IDUA pE-neo (scFab-IDUA) — scFab-IDUA hPPT-1 100 IgG4HC-PPT1 pE-neo (IgG4 HC-PPT1) pE-hygr (LC3) IgG4 PPT1 101 IgG4HC-GS5-PPT1 pE-neo (IgG4 HC-GS5-PPT1) pE-hygr (LC3) IgG4 GS5-PPT1 102IgG4 HC-GS10-PPT1 pE-neo (IgG4 HC-GS10-PPT1) pE-hygr (LC3) IgG4GS10-PPT1 103 IgG4 HC-GS20-PPT1 pE-neo (IgG4 HC-GS20-PPT1) pE-hygr (LC3)IgG4 GS20-PPT1 104 Fab HC-GS3-PPT1 pE-neo (Fab HC-GS3-PPT1) pE-hygr(LC3) Fab GS3-PPT1 105 Tandem Fab HC-PPT1 pE-neo (Tandem Fab HC-PPT1)pE-hygr (LC3) Tandem Fab PPT1 hASM 106 IgG4 HC-ASM pE-neo (IgG4 HC-ASM)pE-hygr (LC3) IgG4 ASM 107 IgG4 HC-GS5-ASM pE-neo (IgG4 HC-GS5-ASM)pE-hygr (LC3) IgG4 GS5-ASM 108 IgG4 HC-GS10-ASM pE-neo (IgG4HC-GS10-ASM) pE-hygr (LC3) IgG4 GS10-ASM 109 IgG4 HC-GS20-ASM pE-neo(IgG4 HC-GS20-ASM) pE-hygr (LC3) IgG4 GS20-ASM 110 Fab HC-GS3-ASM pE-neo(Fab HC-GS3-ASM) pE-hygr (LC3) Fab GS3-ASM 111 Fab HC-GS5-ASM pE-neo(Fab HC-GS5-ASM) pE-hygr (LC3) Fab GS5-ASM 112 Fab HC-GS10-ASM pE-neo(Fab HC-GS10-ASM) pE-hygr (LC3) Fab GS10-ASM 113 Fab HC-GS20-ASM pE-neo(Fab HC-GS20-ASM) pE-hygr (LC3) Fab GS20-ASM 114 Tandem Fab HC-ASMpE-neo (Tandem Fab HC-ASM) pE-hygr (LC3) Tandem Fab ASM hARSA 115 IgG4HC-ARSA pE-neo (IgG4 HC-ARSA) pE-hygr (LC3) IgG4 ARSA 116 IgG4HC-GS5-ARSA pE-neo (IgG4 HC-GS5-ARSA) pE-hygr (LC3) IgG4 GS5-ARSA 117IgG4 HC-GS10-ARSA pE-neo (IgG4 HC-GS10-ARSA) pE-hygr (LC3) IgG4GS10-ARSA 118 IgG4 HC-GS20-ARSA pE-neo (IgG4 HC-GS20-ARSA) pE-hygr (LC3)IgG4 GS20-ARSA 119 Fab HC-GS3-ARSA pE-neo (Fab HC-GS3-ARSA) pE-hygr(LC3) Fab GS3-ARSA 120 Fab HC-GS5-ARSA pE-neo (Fab HC-GS5-ARSA) pE-hygr(LC3) Fab GS5-ARSA 121 Fab HC-GS10-ARSA pE-neo (Fab HC-GS10-ARSA)pE-hygr (LC3) Fab GS10-ARSA 122 Fab HC-GS20-ARSA pE-neo (FabHC-GS20-ARSA) pE-hygr (LC3) Fab GS20-ARSA 123 Tandem Fab HC-ARSA pE-neo(Tandem Fab HC-ARSA) pE-hygr (LC3) Tandem Fab ARSA

TABLE 20(B) List of fusion proteins between various human lysosomalenzymes and humanized anti-hTfR antibody Designation of lysosomalDesignation of Light-chain enzyme-humanized Designation of SEQDesignation of expression expression anti-hTfR antibody lysosome ID NOfusion protein vector vector fusion protein hSGSH 124 IgG4 HC-SGSHpE-neo (IgG4 HC-SGSH) pE-hygr (LC3) IgG4 SGSH 125 IgG4 HC-GS5-SGSHpE-neo (IgG4 HC-GS5-SGSH) pE-hygr (LC3) IgG4 GS5-SGSH 126 IgG4HC-GS10-SGSH pE-neo (IgG4 HC-GS10-SGSH) pE-hygr (LC3) IgG4 GS10-SGSH 127IgG4 HC-GS20-SGSH pE-neo (IgG4 HC-GS20-SGSH) pE-hygr (LC3) IgG4GS20-SGSH 128 Fab HC-GS3-SGSH pE-neo (Fab HC-GS3-SGSH) pE-hygr (LC3) FabGS3-SGSH 129 Tandem Fab HC-SGSH pE-neo (Tandem Fab HC-SGSH) pE-hygr(LC3) TandemFab SGSH hGBA 130 IgG4 HC-GBA pE-neo (IgG4 HC-GBA) pE-hygr(LC3) IgG4 GBA 131 IgG4 HC-GS5-GBA pE-neo (IgG4 HC-GS5-GBA) pE-hygr(LC3) IgG4 GS5-GBA 132 IgG4 HC-GS10-GBA pE-neo (IgG4 HC-GS10-GBA)pE-hygr (LC3) IgG4 GS10-GBA 133 IgG4 HC-GS20-GBA pE-neo (IgG4HC-GS20-GBA) pE-hygr (LC3) IgG4 GS20-GBA 134 Fab HC-GS3-GBA pE-neo (FabHC-GS3-GBA) pE-hygr (LC3) Fab GS3-GBA 135 Tandem Fab HC-GBA pE-neo(Tandem Fab HC-GBA) pE-hygr (LC3) TandemFab GBA hTPP-1 136 IgG4 HC-TPP1pE-neo (IgG4 HC-TPP1) pE-hygr (LC3) IgG4 TPP1 137 IgG4 HC-GS5-TPP1pE-neo (IgG4 HC-GS5-TPP1) pE-hygr (LC3) IgG4 GS5-TPP1 138 IgG4HC-GS10-TPP1 pE-neo (IgG4 HC-GS10-TPP1) pE-hygr (LC3) IgG4 GS10-TPP1 139IgG4 HC-GS20-TPP1 pE-neo (IgG4 HC-GS20-TPP1) pE-hygr (LC3) IgG4GS20-TPP1 140 Fab HC-GS3-TPP1 pE-neo (Fab HC-GS3-TPP1) pE-hygr (LC3) FabGS3-TPP1 141 Tandem Fab HC-TPP1 pE-neo (TandemFab HC-TPP1) pE-hygr (LC3)Tandem Fab TPP1 hNAGLU 142 IgG4 HC-NAGLU pE-neo (IgG4 HC-NAGLU) pE-hygr(LC3) IgG4 NAGLU 143 IgG4 HC-GS5-NAGLU pE-neo (IgG4 HC-GS5-NAGLU)pE-hygr (LC3) IgG4 GS5-NAGLU 144 IgG4 HC-GS10-NAGLU pE-neo (IgG4HC-GS10-NAGLU) pE-hygr (LC3) IgG4 GS10-NAGLU 145 IgG4 HC-GS20-NAGLUpE-neo (IgG4 HC-GS20-NAGLU) pE-hygr (LC3) IgG4 GS20-NAGLU 146 FabHC-GS3-NAGLU pE-neo (Fab HC-GS3-NAGLU) pE-hygr (LC3) Fab GS3-NAGLU 147Fab HC-GS5-NAGLU pE-neo (Fab HC-GS5-NAGLU) pE-hygr (LC3) Fab GS5-NAGLU148 Fab HC-GS10-NAGLU pE-neo (Fab HC-GS10-NAGLU) pE-hygr (LC3) FabGS10-NAGLU 149 Fab HC-GS20-NAGLU pE-neo (Fab HC-GS20-NAGLU) pE-hygr(LC3) Fab GS20-NAGLU hGUSB 150 IgG4 HC-GUSB pE-neo (IgG4 HC-GUSB)pE-hygr (LC3) IgG4 GUSB 151 IgG4 HC-GS5-GUSB pE-neo (IgG4 HC-GS5-GUSB)pE-hygr (LC3) IgG4 GS5-GUSB 152 IgG4 HC-GS10-GUSB pE-neo (IgG4HC-GS10-GUSB) pE-hygr (LC3) IgG4 GS10-GUSB 153 IgG4 HC-GS20-GUSB pE-neo(IgG4 HC-GS20-GUSB) pE-hygr (LC3) IgG4 GS20-GUSB 154 Fab HC-GS3-GUSBpE-neo (Fab HC-GS3-GUSB) pE-hygr (LC3) Fab GS3-GUSB 155 Fab HC-GS5-GUSBpE-neo (Fab HC-GS5-GUSB) pE-hygr (LC3) Fab GS5-GUSB 156 Fab HC-GS10-GUSBpE-neo (Fab HC-GS10-GUSB) pE-hygr (LC3) Fab GS10-GUSB 157 FabHC-GS20-GUSB pE-neo (Fab HC-GS20-GUSB) pE-hygr (LC3) Fab GS20-GUSB

TABLE 20(C) List of fusion proteins between various human lysosomalenzymes and humanized anti-hTfR antibody Designation of lysosomalDesignation of Light-chain enzyme-humanized Designation of SEQDesignation of expression expression anti-hTfR antibody lysosome ID NOfusion protein vector vector fusion protein hGALC 158 IgG4 HC-GALCpE-neo (IgG4 HC-GALC) pE-hygr (LC3) IgG4 GALC 159 IgG4 HC-GS5-GALCpE-neo (IgG4 HC-GS5-GALC) pE-hygr (LC3) IgG4 GS5-GALC 160 IgG4HC-GS10-GALC pE-neo (IgG4 HC-GS10-GALC) pE-hygr (LC3) IgG4 GS10-GALC 161IgG4 HC-GS20-GALC pE-neo (IgG4 HC-GS20-GALC) pE-hygr (LC3) IgG4GS20-GALC 162 Fab HC-GS3-GALC pE-neo (Fab HC-GS3-GALC) pE-hygr (LC3) FabGS3-GALC 163 Fab HC-GS5-GALC pE-neo (Fab HC-GS5-GALC) pE-hygr (LC3) FabGS5-GALC 164 Fab HC-GS10-GALC pE-neo (Fab HC-GS10-GALC) pE-hygr (LC3)Fab GS10-GALC 165 Fab HC-GS20-GALC pE-neo (Fab HC-GS20-GALC) pE-hygr(LC3) Fab GS20-GALC hAC 166 IgG4 HC-AC pE-neo (IgG4 HC-AC) pE-hygr (LC3)IgG4 AC 167 IgG4 HC-GS5-AC pE-neo (IgG4 HC-GS5-AC) pE-hygr (LC3) IgG4GS5-AC 168 IgG4 HC-GS10-AC pE-neo (IgG4 HC-GS10-AC) pE-hygr (LC3) IgG4GS10-AC 169 IgG4 HC-GS20-AC pE-neo (IgG4 HC-GS20-AC) pE-hygr (LC3) IgG4GS20-AC 170 Fab HC-GS3-AC pE-neo (Fab HC-GS3-AC) pE-hygr (LC3) FabGS3-AC 171 Fab HC-GS5-AC pE-neo (Fab HC-GS5-AC) pE-hygr (LC3) Fab GS5-AC172 Fab HC-GS10-AC pE-neo (Fab HC-GS10-AC) pE-hygr (LC3) Fab GS10-AC 173Fab HC-GS20-AC pE-neo (Fab HC-GS20-AC) pE-hygr (LC3) Fab GS20-AC hFUCAl174 IgG4 HC-FUCA1 pE-neo (IgG4 HC-FUCA1) pE-hygr (LC3) IgG4 FUCA1 175IgG4 HC-GS5-FUCA1 pE-neo (IgG4 HC-GS5-FUCA1) pE-hygr (LC3) IgG4GS5-FUCA1 176 IgG4 HC-GS10-FUCA1 pE-neo (IgG4 HC-GS10-FUCA1) pE-hygr(LC3) IgG4 GS10-FUCA1 177 IgG4 HC-GS20-FUCA1 pE-neo (IgG4 HC-GS20-FUCA1)pE-hygr (LC3) IgG4 GS20-FUCA1 178 Fab HC-GS3-FUCA1 pE-neo (FabHC-GS3-FUCA1) pE-hygr (LC3) Fab GS3-FUCA1 179 Fab HC-GS5-FUCA1 pE-neo(Fab HC-GS5-FUCA1) pE-hygr (LC3) Fab GS5-FUCA1 180 Fab HC-GS10-FUCA1pE-neo (Fab HC-GS10-FUCA1) pE-hygr (LC3) Fab GS10-FUCA1 181 FabHC-GS20-FUCA1 pE-neo (Fab HC-GS20-FUCA1) pE-hygr (LC3) Fab GS20-FUCA1hLAMAN 182 IgG4 HC-LAMAN pE-neo (IgG4 HC-LAMAN) pE-hygr (LC3) IgG4 LAMAN183 IgG4 HC-GS5-LAMAN pE-neo (IgG4 HC-GS5-LAMAN) pE-hygr (LC3) IgG4GS5-LAMAN 184 IgG4 HC-GS10-LAMAN pE-neo (IgG4 HC-GS10-LAMAN) pE-hygr(LC3) IgG4 GS10-LAMAN 185 IgG4 HC-GS20-LAMAN pE-neo (IgG4 HC-GS20-LAMAN)pE-hygr (LC3) IgG4 GS20-LAMAN 186 Fab HC-GS3-LAMAN pE-neo (FabHC-GS3-LAMAN) pE-hygr (LC3) Fab GS3-LAMAN 187 Fab HC-GS5-LAMAN pE-neo(Fab HC-GS5-LAMAN) pE-hygr (LC3) Fab GS5-LAMAN 188 Fab HC-GS10-LAMANpE-neo (Fab HC-GS10-LAMAN) pE-hygr (LC3) Fab GS10-LAMAN 189 FabHC-GS20-LAMAN pE-neo (Fab HC-GS20-LAMAN) pE-hygr (LC3) Fab GS20-LAMAN

Example 34 Measurement of Affinity of Fusion Proteins Between VariousHuman Lysosomal Enzymes and Humanized Anti-hTfR Antibody to Human TfRand Monkey TfR, and Enzymatic Activity of the Fusion Proteins

The affinity to human TfR and monkey TfR was measured by the methoddescribed in Example 7. The activity of the human lysosomal enzyme wasmeasured by the method described below. However, the enzymatic activityof hAC and hLAMAN was not measured.

Measurement of Activity of hIDUA

The purified anti-TfR antibody fusion protein of hIDUA was diluted to anappropriate concentration with a 50 mM citrate buffer solution (pH: 3.5)containing 0.1% BSA, and 20 μL of the resulting dilution was added toeach well of FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone(4MU) (Sigma Inc.) was used as a standard. Next, as a substrate,4-Methylumbelliferyl α-L-Iduronide (glycosynth Inc.) was diluted to 1 mMwith the above-described buffer solution, and 60 μL of the resultingdilution was added to each well. The mixture was stirred by a platemixer, and then reacted at 37° C. for 1 hour. After the reaction, 200 μLof a 0.05 M Glycine/NaOH buffer solution (pH: 10.6) was added to eachwell to stop the reaction, and measurement was performed at anexcitation wavelength of 365 nm and a detection wavelength of 460 nm bya plate reader. The enzymatic activity was determined in terms of aspecific activity per protein unit weight (mg) with one unit defined as4 MU μmol/minute at 37° C. For the following fusion proteins, thespecific activity was determined as well.

Measurement of Activity of hPPT-1:

β-Glucosidase (Oriental Yeast Co., Ltd.) was diluted to 0.25 mg/mL witha 0.4 M Na₂HPO₄/0.2 M citrate buffer solution (pH: 4.0) containing 0.2%BSA and 0.00625% Triton X-100, and 20 μL of the resulting dilution wasadded to each well of FluoroNunc Plate F96 (Nunc Inc.). Next, thepurified anti-TfR antibody fusion protein of hPPT-1 was diluted to anappropriate concentration with the above-described buffer solution, and20 μL of the resulting dilution was added to each well.4-Methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next,as a substrate, 4-Methylumbelliferyl6-Thio-palmitate-β-D-glucopyranoside (Santa Cruz Biotechnology Inc.) wasdiluted to 0.25 mM with the above-described buffer solution, and 20 μLof the resulting dilution was added to each well. The mixture wasstirred by a plate mixer, and then reacted at 37° C. for 1 hour. Afterthe reaction, 150 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6)was added to each well to stop the reaction, and measurement wasperformed at an excitation wavelength of 365 nm and a detectionwavelength of 460 nm by a plate reader. The enzymatic activity wasdetermined with one unit defined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hASM:

The purified anti-TfR antibody fusion protein of hASM was diluted to anappropriate concentration with a 250 mM acetate buffer solution (pH:5.0) containing 0.1 mM zinc acetate, 0.25 mg/mL BSA and 0.15% Tween 20,and 20 μL of the resulting dilution was added to each well of FluoroNuncPlate F96 (Nunc Inc.). 4-Methylumbelliferone (4MU) (Sigma Inc.) was usedas a standard. Next, as a substrate,6-Hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (CarbosynthInc.) was diluted to 1 mM with the above-described buffer solution, and20 μL of the resulting dilution was added to each well. The mixture wasstirred by a plate mixer, and then reacted at 37° C. for 1 hour. Afterthe reaction, 200 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6)was added to each well to stop the reaction, and measurement wasperformed at an excitation wavelength of 365 nm and a detectionwavelength of 460 nm by a plate reader. The enzymatic activity wasdetermined in terms of a specific activity per protein unit weight (mg)with one unit defined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hARSA:

The purified anti-TfR antibody fusion protein of hARSA was diluted to anappropriate concentration with a 50 mM acetate buffer solution (pH: 5.0)containing 0.5% BSA and 1.0% Triton-X 100, and 20 μL of the resultingdilution was added to each well of FluoroNunc Plate F96 (Nunc Inc.).4-Methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next,as a substrate, 4-methylumbelliferyl sulfate potassium salt (Sigma Inc.)was diluted to 5 mM with the above-described buffer solution, and 100 μLof the resulting dilution was added to each well. The mixture wasstirred by a plate mixer, and then reacted at 37° C. for 1 hour. Afterthe reaction, 150 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6)was added to each well to stop the reaction, and measurement wasperformed at an excitation wavelength of 365 nm and a detectionwavelength of 460 nm by a plate reader. The enzymatic activity wasdetermined with one unit defined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hSGSH:

The purified anti-TfR antibody fusion protein of hSGSH was diluted to anappropriate concentration with a 50 mM acetate buffer solution (pH: 5.0)containing 0.2% BSA, and 20 μL of the resulting dilution was added toeach well of FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone(4MU) (Sigma Inc.) was used as a standard. Next, as a substrate,4-methylumbelliferyl 2-deoxy-2-sulfamino-α-D-glucopyranoside (CarbosynthInc.) was diluted to 5 mM with the above-described buffer solution, and20 μL of the resulting dilution was added to each well. The mixture wasstirred by a plate mixer, and then reacted at 37° C. for 2 hours. Afterthe reaction, 0.5 mg/mL of α-Glucosidase (Oriental Yeast Co., Ltd.) wasdiluted with the above-described buffer solution, 20 μL of the resultingdilution was added to each well, and the mixture was reacted at 37° C.for 17 hours. After the reaction, 150 μL of a 0.05 M Glycine/NaOH buffersolution (pH: 10.6) was added to each well to stop the reaction, andmeasurement was performed at an excitation wavelength of 365 nm and adetection wavelength of 460 nm by a plate reader. The enzymatic activitywas determined with one unit defined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hGBA:

The purified anti-TfR antibody fusion protein of hGBA was diluted to anappropriate concentration with a 100 mM phosphate buffer solution (pH:6.0) containing 0.1% BSA, 0.15% Triton-X 100 and 0.125% sodiumtaurocholate, and 20 μL of the resulting dilution was added to each wellof FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone (4MU) (SigmaInc.) was used as a standard. Next, as a substrate, 4-methylumbelliferylβ-D-glucopyranoside (Sigma Inc.) was diluted to 3.5 mM with theabove-described buffer solution, and 70 μL of the resulting dilution wasadded to each well. The mixture was stirred by a plate mixer, and thenreacted at 37° C. for 1 hour. After the reaction, 150 μL of a 0.05 MGlycine/NaOH buffer solution (pH: 10.6) was added to each well to stopthe reaction, and measurement was performed at an excitation wavelengthof 365 nm and a detection wavelength of 460 nm by a plate reader. Theenzymatic activity was determined with one unit defined as 4 MUμmol/minute at 37° C.

Measurement of Activity of hTPP-1:

The purified anti-TfR antibody fusion protein of hTPP-1 was diluted toan appropriate concentration with a 50 mM sodium formate buffer solution(pH: 3.5) containing 150 mM NaCl and 0.1% Triton-X 100, and theresulting dilution was preincubated at 37° C. for 1 hour.7-Amino-4-methylcoumarin (AMC) (Sigma Inc.) was used as a standard, andsimilarly treated. After 1 hour, 20 μL of the dilution was added to eachwell of FluoroNunc Plate F96 (Nunc Inc.). Next, as a substrate,Ala-Ala-Phe-7-amido-4-methylcoumarin (Sigma Inc.) was diluted to 25 mMwith a 100 mM acetate buffer solution (pH: 4.0) containing 150 mM NaCland 0.1% Triton-X 100, and 40 μL of the resulting dilution was added toeach well. The mixture was stirred by a plate mixer, and then reacted at37° C. for 1 hour. After the reaction, 150 μL of a 100 mMmonochloroacetate/130 mM NaOH/100 mM acetate buffer solution (pH: 4.3)was added to each well to stop the reaction, and measurement wasperformed at an excitation wavelength of 365 nm and a detectionwavelength of 460 nm by a plate reader. The enzymatic activity wasdetermined with one unit defined as AMC μmol/minute at 37° C.

Measurement of Activity of hGAA:

The purified anti-TfR antibody fusion protein of hGAA was diluted to anappropriate concentration with PBS (−) (pH: 7.2) containing 0.5% BSA,and 20 μL of the resulting dilution was added to each well of FluoroNuncPlate F96 (Nunc Inc.). 4-Methylumbelliferone (4MU) (Sigma Inc.) was usedas a standard. Next, as a substrate, 4-methylumbelliferyla-D-glucopyranoside (Sigma Inc.) was diluted to 2.2 mM with a 200 mMacetate buffer solution (pH: 4.3), and 20 μL of the resulting dilutionwas added to each well. The mixture was stirred by a plate mixer, andthen reacted at 37° C. for 1 hour. After the reaction, 200 μL of a 0.05M Glycine/NaOH buffer solution (pH: 10.6) was added to each well to stopthe reaction, and measurement was performed at an excitation wavelengthof 365 nm and a detection wavelength of 460 nm by a plate reader. Theenzymatic activity was determined with one unit defined as 4 MUμmol/minute at 37° C.

Measurement of Activity of hNAGLU:

The purified anti-TfR antibody fusion protein of hNAGLU was diluted toan appropriate concentration with a 50 mM acetate buffer solution (pH:4.3) containing 0.1% BSA, and 20 μL of the resulting dilution was addedto each well of FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone(4MU) (Sigma Inc.) was used as a standard. Next, as a substrate,4-methylumbelliferyl N-acetyl-α-D-glucosaminide (Calbiochem) was dilutedto 1 mM with the above-described buffer solution, and 20 μL of theresulting dilution was added to each well. The mixture was stirred by aplate mixer, and then reacted at 37° C. for 1 hour. After the reaction,150 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6) was added toeach well to stop the reaction, and measurement was performed at anexcitation wavelength of 365 nm and a detection wavelength of 460 nm bya plate reader. The enzymatic activity was determined with one unitdefined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hGUSB:

The purified anti-TfR antibody fusion protein of hGUSB was diluted to anappropriate concentration with a 25 mM acetate buffer solution (pH: 4.5)containing 50 mM NaCl, and 20 μL of the resulting dilution was added toeach well of FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone(4MU) (Sigma Inc.) was used as a standard. Next, as a substrate,4-methylumbelliferyl-β-D-glucuronide hydrate (Sigma Inc.) was diluted to2 mM with the above-described buffer solution, and 20 μL of theresulting dilution was added to each well. The mixture was stirred by aplate mixer, and then reacted at 37° C. for 1 hour. After the reaction,150 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6) was added toeach well to stop the reaction, and measurement was performed at anexcitation wavelength of 365 nm and a detection wavelength of 460 nm bya plate reader. The enzymatic activity was determined with one unitdefined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hGALC:

The purified anti-TfR antibody fusion protein of GALC was diluted to anappropriate concentration with a 50 mM acetate buffer solution (pH: 4.5)containing 125 mM NaCl and 0.5% Triton X-100, and 20 μL of the resultingdilution was added to each well of FluoroNunc Plate F96 (Nunc Inc.).4-Methylumbelliferone (4MU) (Sigma Inc.) was used as a standard. Next,as a substrate, 4-methylumbelliferyl-β-D-galactopyranoside (Sigma Inc.)was diluted to 1 mM with the above-described buffer solution, and 20 μLof the resulting dilution was added to each well. The mixture wasstirred by a plate mixer, and then reacted at 37° C. for 1 hour. Afterthe reaction, 150 μL of a 0.05 M Glycine/NaOH buffer solution (pH: 10.6)was added to each well to stop the reaction, and measurement wasperformed at an excitation wavelength of 365 nm and a detectionwavelength of 460 nm by a plate reader. The enzymatic activity wasdetermined with one unit defined as 4 MU μmol/minute at 37° C.

Measurement of Activity of hFUCA1:

The purified anti-TfR antibody fusion protein of FUCA1 was diluted to anappropriate concentration with a 100 mM citrate buffer solution (pH:5.5) containing 0.2% BSAI, and 20 μL of the resulting dilution was addedto each well of FluoroNunc Plate F96 (Nunc Inc.). 4-Methylumbelliferone(4MU) (Sigma Inc.) was used as a standard. Next, as a substrate,4-MU-α-L-fucopyranoside (Sigma Inc.) was diluted to 2 mM with theabove-described buffer solution, and 20 μL of the resulting dilution wasadded to each well. The mixture was stirred by a plate mixer, and thenreacted at 37° C. for 1 hour. After the reaction, 150 μL of a 0.05 MGlycine/NaOH buffer solution (pH: 10.6) was added to each well to stopthe reaction, and measurement was performed at an excitation wavelengthof 365 nm and a detection wavelength of 460 nm by a plate reader. Theenzymatic activity was determined with one unit defined as 4 MUμmol/minute at 37° C.

Table 21 shows the measurements of the affinity of fusion proteinsbetween various human lysosomal enzymes and a humanized anti-hTfRantibody to human TfR and monkey TfR, and the enzymatic activity of thefusion proteins. The affinity and the enzymatic activity were measuredonly for some of the fusion proteins between lysosomal enzymes and ahumanized anti-hTfR antibody which had been obtained in Example 33.Specifically, the affinity to human TfR and monkey TfR was measured for24 types of fusion proteins, and the enzymatic activity was measured for20 types of fusion proteins.

The fusion proteins whose enzymatic activity had been measured each hadan enzymatic activity as a lysosomal enzyme. Further, the fusionproteins whose affinity to human TfR had been measured each had a highaffinity to human TfR, even those having the lowest affinity showed aK_(D) value of 2.09×10⁻¹⁰, and 19 types of fusion proteins showed aK_(D) value of lower than 1.00×10⁻¹². Further, the fusion proteins whoseaffinity to monkey TfR had been measured each had a high affinity tomonkey TfR, even those having the lowest affinity showed a K_(D) valueof 3.05×10⁻⁸, and 12 types of fusion proteins showed a K_(D) value oflower than 1.00×10⁻¹².

The anti-hTfR antibody used here is one whose heavy chain is a heavychain (IgG) having as a variable region an amino acid sequence set forthas SEQ ID NO: 65, or a Fab heavy chain, and whose light chain has as avariable region an amino acid sequence set forth as SEQ ID NO: 18. Theseresults indicate that by bringing a lysosomal enzyme into a form of afusion protein between the lysosomal enzyme and an antibody comprisingthose amino acid sequences as variable regions, the lysosomal enzyme canbe allowed to pass through BBB via human TfR and exhibit an enzymaticactivity in the brain.

TABLE 21 Affinity of fusion proteins between various human lysosomalenzymes and humanized anti- hTfR antibody to human TfR and monkey TfR,and enzymatic activity of the fusion proteins Designation of lysosomalSpecific enzyme-humanized enzymatic anti-hTfR antibody activity Affinityto human TfR and monkey TfR fusion protein (Unit/mg) Type kon (1/Ms)koff (1/s) K_(D) (M) Fab-GS3-GAA 4.81 Human TfR 5.95 × 10⁵  3.39 × 10⁻⁴ 2.09 × 10⁻¹⁰ Monkey TfR 4.73 × 10⁵  1.45 × 10⁻²  3.05 × 10⁻⁵ IgG4-IDUA1.55 Human TfR 9.80 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 5.61 ×10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² IgG4-GS20-IDUA 1.37 Human TfR 9.24 × 10⁵<1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 4.81 × 10⁵ <1.00 × 10⁻⁷ <1.00 ×10⁻¹² Fab-GS3-IDUA 1.7 Human TfR 2.93 × 10⁵  5.28 × 10⁻⁵  1.08 × 10⁻¹¹Monkey TfR 2.21 × 10⁵  2.92 × 10⁻⁴  1.32 × 10⁻² IgG4-PPT1 9.4 Human TfR7.37 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 3.07 × 10⁵ <1.00 × 10⁻⁷<1.00 × 10⁻¹² Fab-GS3-PPT1 9.1 Human TfR 4.17 × 10⁵ <1.00 × 10⁻⁷ <1.00 ×10⁻¹² Monkey TfR 9.53 × 10⁴  9.13 × 10⁻⁴  1.32 × 10⁻² Fab-GS3-ASM 0.105Human TfR 5.26 × 10⁵  3.63 × 10⁻⁵  6.89 × 10⁻¹¹ Monkey TfR 2.74 × 10⁵ 5.49 × 10⁻⁴  2.00 × 10⁻² IgG4-GS10-ARSA 0.72 Human TfR 4.60 × 10⁵ <1.00× 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 1.69 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹²Fab-GS3-ARSA 0.55 Human TfR 6.70 × 10⁵  4.02 × 10⁻⁵  6.00 × 10⁻¹² MonkeyTfR 2.58 × 10⁵  2.81 × 10⁻⁴  1.09 × 10⁻² Fab-GS3-SGSH 0.068 Human TfR7.21 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 3.88 × 10⁵ <1.00 × 10⁻⁷<1.00 × 10⁻¹² IgG4-GS10-GBA 7.0 Human TfR 9.57 × 10⁵ <1.00 × 10⁻⁷ <1.00× 10⁻¹² Monkey TfR 4.49 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Fab-GS3-GBA 9.2Human TfR 6.23 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 2.99 × 10⁵ 1.42 × 10⁻⁵  4.75 × 10⁻² IgG4-TPP1 0.26 Human TfR 7.58 × 10⁵ <1.00 ×10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 3.41 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹²IgG4-GS20-TPP1 0.21 Human TfR 6.82 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹²Monkey TfR 3.02 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Fab-GS3-NAGLU Notmeasured Human TfR 2.91 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 1.54× 10⁵  4.02 × 10⁻⁴  2.62 × 10⁻² Fab-GS3-GUSB 8.49 Human TfR 4.49 × 10⁵<1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 2.50 × 10⁵ <1.00 × 10⁻⁷ <1.00 ×10⁻¹² IgG4-GS10-GALC 0.35 Human TfR 3.14 × 10⁴ <1.00 × 10⁻⁷ <1.00 ×10⁻¹² Monkey TfR 2.80 × 10⁴ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Fab-GS3-GALC 1.1Human TfR 2.24 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 1.11 × 10⁵ 1.54 × 10⁻⁴  1.40 × 10⁻² IgG4-GS10-AC Not measured Human TfR 5.76 × 10⁵<1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 3.01 × 10⁵ <1.00 × 10⁻⁷ <1.00 ×10⁻¹² Fab-GS3-AC Not measured Human TfR 5.01 × 10⁵  1.05 × 10⁻⁴  2.09 ×10⁻¹⁰ Monkey TfR 2.77 × 10⁵  3.27 × 10⁻⁵  1.18 × 10⁻⁵ IgG4-GS10-FUCA124.2 Human TfR 4.66 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 2.46 ×10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹² Fab-GS3-FUCA1 27.6 Human TfR 2.82 × 10⁵<1.00 × 10⁻⁷ <1.00 × 10⁻¹² Monkey TfR 1.51 × 10⁵  4.88 × 10⁻⁵  3.23 ×10⁻¹⁰ Fab-GS3-LAMAN Not measured Human TfR 7.29 × 10⁵ <1.00 × 10⁻⁷ <1.00× 10⁻¹² Monkey TfR 3.57 × 10⁵ <1.00 × 10⁻⁷ <1.00 × 10⁻¹²

INDUSTRIAL APPLICABILITY

The anti-hTfR antibody of the present invention, when fused withphysiologically active proteins, low-molecular-weight compounds and thelike of interest, can make them able to pass through the blood-brainbarrier, and is, therefore, highly useful in providing a means todeliver physiologically active proteins to the brain,low-molecular-weight compounds and the like which are needed to act inthe central nervous system.

REFERENCE SIGNS LIST

1 Blood vessel

2 Brain parenchyma

3 Nerve-like cells

4 Purkinje cells

SEQUENCE LISTING FREE TEXT

SEQ ID NO:3: Amino acid sequence of exemplified linker 1

SEQ ID NO:4: Amino acid sequence of exemplified linker 2

SEQ ID NO:5: Amino acid sequence of exemplified linker 3

SEQ ID NO:6: Amino acid sequence 1 of CDR1 in the light chain of mouseanti-hTfR antibody No.3

SEQ ID NO:7: Amino acid sequence 2 of CDR1 in the light chain of mouseanti-hTfR antibody No.3

SEQ ID NO:8: Amino acid sequence 1 of CDR2 in the light chain of mouseanti-hTfR antibody No.3

SEQ ID NO:9: Amino acid sequence 2 of CDR2 in the light chain of mouseanti-hTfR antibody No.3

SEQ ID NO:10: Amino acid sequence of CDR3 in the light chain of mouseanti-hTfR antibody No.3

SEQ ID NO:11: Amino acid sequence 1 of CDR1 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:12: Amino acid sequence 2 of CDR1 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:13: Amino acid sequence 1 of CDR2 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:14: Amino acid sequence 2 of CDR2 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:15: Amino acid sequence 1 of CDR3 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:16: Amino acid sequence 2 of CDR3 in the heavy chain of mouseanti-hTfR antibody No.3

SEQ ID NO:17: Amino acid sequence 1 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:18: Amino acid sequence 2 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:19: Amino acid sequence 3 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:20: Amino acid sequence 4 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:21: Amino acid sequence 5 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:22: Amino acid sequence 6 of the light chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:23: Amino acid sequence of the light chain of humanizedanti-hTfR antibody No.3 containing amino acid sequence 2 as the variableregion

SEQ ID NO:24: Nucleotide sequence encoding the amino acid sequence ofthe light chain of humanized anti-hTfR antibody No.3 containing aminoacid sequence 2 as the variable region, synthetic sequence

SEQ ID NO:25: Amino acid sequence of the light chain of humanizedanti-hTfR antibody No.3 containing amino acid sequence 4 as the variableregion

SEQ ID NO:26: Nucleotide sequence encoding the amino acid sequence ofthe light chain of humanized anti-hTfR antibody No.3 containing aminoacid sequence 4 as the variable region, synthetic sequence

SEQ ID NO:27: Amino acid sequence of the light chain of humanizedanti-hTfR antibody No.3 containing amino acid sequence 5 as the variableregion

SEQ ID NO:28: Nucleotide sequence encoding the amino acid sequence ofthe light chain of humanized anti-hTfR antibody No.3 containing aminoacid sequence 5 as the variable region, synthetic sequence

SEQ ID NO:29: Amino acid sequence of the light chain of humanizedanti-hTfR antibody No.3 containing amino acid sequence 6 as the variableregion

SEQ ID NO:30: Nucleotide sequence encoding the amino acid sequence ofthe light chain of humanized anti-hTfR antibody No.3 containing aminoacid sequence 6 as the variable region, synthetic sequence

SEQ ID NO:31: Amino acid sequence 1 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:32: Amino acid sequence 2 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:33: Amino acid sequence 3 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:34: Amino acid sequence 4 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:35: Amino acid sequence 5 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:36: Amino acid sequence 6 of the heavy chain variable regionof humanized anti-hTfR antibody No.3

SEQ ID NO:37: Amino acid sequence of the heavy chain of humanizedanti-hTfR antibody No.3 containing amino acid sequence 2 as the variableregion

SEQ ID NO:38: Nucleotide sequence encoding the amino acid sequence ofthe heavy chain of humanized anti-hTfR antibody No.3 containing aminoacid sequence 2 as the variable region, synthetic sequence

SEQ ID NO:39: Amino acid sequence of the heavy chain (IgG4) of humanizedanti-hTfR antibody No.3 containing amino acid sequence 2 as the variableregion

SEQ ID NO:40: Nucleotide sequence encoding the amino acid sequence ofthe heavy chain (IgG4) of humanized anti-hTfR antibody No.3 containingamino acid sequence 2 as the variable region, synthetic sequence

SEQ ID NO:41: Primer hTfR5′, synthetic sequence

SEQ ID NO:42: Primer hTfR3′, synthetic sequence

SEQ ID NO:43: Primer Hyg-Sfi5′, synthetic sequence

SEQ ID NO:44: Primer Hyg-BstX3′, synthetic sequence

SEQ ID NO:45: Nucleotide sequence of the DNA in which a neomycinresistance gene flanked by loxP sequences was placed on the cDNA's 3′side of a cDNA encoding chimeric hTfR, synthetic sequence

SEQ ID NO:46: 5′-arm sequence of targeting vector, synthetic sequence

SEQ ID NO:47: 3′-arm sequence of targeting vector, synthetic sequence

SEQ ID NO:48: Amino acid sequence of the light chain variable region ofmouse anti-hTfR antibody No.3

SEQ ID NO:49: Amino acid sequence of the heavy chain variable region ofmouse anti-hTfR antibody No.3

SEQ ID NO:51: Amino acid sequence of fusion protein of heavy chain ofanti-hTfR antibody No.3 (humanized 2) and hI2S, synthetic sequence

SEQ ID NO:52: Nucleotide sequence encoding the amino acid sequence offusion protein of heavy chain of anti-hTfR antibody No.3 (humanized 2)and hI2S, synthetic sequence

SEQ ID NO: 53: Amino acid sequence of the fusion protein of heavy chainof humanized anti-hTfR antibody No. 3N and hI2S

SEQ ID NO: 54: Nucleotide sequence encoding the amino acid sequence offusion protein of heavy chain of humanized anti-hTfR antibody No. 3N andhI2S, synthetic sequence

SEQ ID NO: 57: Amino acid sequence of the fusion protein of heavy chainof humanized anti-hTfR antibody No. 3N and hGAA

SEQ ID NO: 58: Amino acid sequence of the fusion protein of heavy chain(IgG4) of humanized anti-hTfR antibody No. 3N and hGAA

SEQ ID NO: 59: Nucleotide sequence encoding the amino acid sequence offusion protein of heavy chain (IgG4) of humanized anti-hTfR antibody No.3N and hGAA, synthetic sequence

SEQ ID NO: 60: Amino acid sequence of single-chain humanized anti-hTfRantibody No. 3N

SEQ ID NO: 61: Amino acid sequence of the Fab heavy chain of humanizedanti-hTfR antibody No. 3N

SEQ ID NO: 62: Amino acid sequence 1 of CDR1 in the heavy chain ofhumanized anti-hTfR antibody No. 3N

SEQ ID NO: 63: Amino acid sequence 2 of CDR1 in the heavy chain ofhumanized anti-hTfR antibody No. 3N

SEQ ID NO: 64: Amino acid sequence of framework region 3 in the heavychain of humanized anti-hTfR antibody No. 3N

SEQ ID NO: 65: Amino acid sequence of the heavy chain variable region ofhumanized anti-hTfR antibody No. 3N

SEQ ID NO: 66: Amino acid sequence of the heavy chain of humanizedanti-hTfR antibody No. 3N

SEQ ID NO: 67: Nucleotide sequence encoding the amino acid sequence ofthe heavy chain of humanized anti-hTfR antibody No. 3N, syntheticsequence

SEQ ID NO: 68: Amino acid sequence of the heavy chain (IgG4) ofhumanized anti-hTfR antibody No. 3N

SEQ ID NO: 69: Nucleotide sequence encoding the amino acid sequence ofthe heavy chain (IgG4) of humanized anti-hTfR antibody No. 3N, syntheticsequence

SEQ ID NO: 70: One example of the amino acid sequence of the Fc regionof human IgG

SEQ ID NO: 71: Amino acid sequence of human IgG Fc region-added Fabheavy chain of humanized anti-hTfR antibody 3N

SEQ ID NO: 72: Nucleotide sequence encoding the amino acid sequence ofhuman IgG Fc region-added Fab heavy chain of humanized anti-hTfRantibody 3N, synthetic sequence

SEQ ID NO: 73: Amino acid sequence of framework region 3 in the heavychain of humanized anti-hTfR antibody No. 3

SEQ ID NO: 74: Amino acid sequence of albumin-binding domain

SEQ ID NO: 75: Amino acid sequence 1 of human IDUA

SEQ ID NO: 76: Amino acid sequence 2 of human IDUA

SEQ ID NO: 77: Amino acid sequence of human PPT-1

SEQ ID NO: 78: Amino acid sequence of human ASM

SEQ ID NO: 79: Amino acid sequence of human ARSA

SEQ ID NO: 80: Amino acid sequence of human SGSH

SEQ ID NO: 81: Amino acid sequence of human GBA

SEQ ID NO: 82: Amino acid sequence of human TPP-1

SEQ ID NO: 83: Amino acid sequence of human NAGLU

SEQ ID NO: 84: Amino acid sequence of human GUSB

SEQ ID NO: 85: Amino acid sequence of human GALC

SEQ ID NO: 86: Amino acid sequence of human AC

SEQ ID NO: 87: Amino acid sequence of human FUCA1

SEQ ID NO: 88: Amino acid sequence of human LAMAN

SEQ ID NO: 89: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGAA, FabHC-GS3-GAA

SEQ ID NO: 90: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hIDUA, IgG4HC-IDUA

SEQ ID NO: 91: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hIDUA, IgG4HC-GS8-IDUA

SEQ ID NO: 92: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hIDUA, IgG4HC-G520-IDUA

SEQ ID NO: 93: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hIDUA, FabHC-GS3-IDUA

SEQ ID NO: 94: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hIDUA, FabHC-GS5-IDUA

SEQ ID NO: 95: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hIDUA, FabHC-GS10-IDUA

SEQ ID NO: 96: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hIDUA, FabHC-G520-IDUA

SEQ ID NO: 97: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hIDUA, Tandem FabHC-IDUA

SEQ ID NO: 98: Amino acid sequence of single-chain humanized anti-hTfRantibody No. 3N(2)

SEQ ID NO: 99: Amino acid sequence of fusion protein betweensingle-chain humanized anti-hTfR antibody No. 3N and IDUA, scFab-IDUA

SEQ ID NO: 100: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4HC-PPT1

SEQ ID NO: 101: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4HC-GS5-PPT1

SEQ ID NO: 102: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4HC-GS10-PPT1

SEQ ID NO: 103: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hPPT-1, IgG4HC-GS20-PPT1

SEQ ID NO: 104: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hPPT-1, FabHC-GS3-PPT1

SEQ ID NO: 105: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hPPT-1, TandemFab HC-PPT1

SEQ ID NO: 106: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hASM, IgG4HC-ASM

SEQ ID NO: 107: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hASM, IgG4HC-GS5-ASM SEQ ID NO: 108: Amino acid sequence of fusion protein betweenthe heavy chain (IgG4) of humanized anti-hTfR antibody No. 3N and hASM,IgG4 HC-GS10-ASM

SEQ ID NO: 109: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hASM, IgG4HC-GS20-ASM

SEQ ID NO: 110: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hASM, FabHC-GS3-ASM

SEQ ID NO: 111: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hASM, FabHC-GS5-ASM

SEQ ID NO: 112: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hASM, FabHC-GS10-ASM

SEQ ID NO: 113: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hASM, FabHC-GS20-ASM

SEQ ID NO: 114: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hASM, Tandem FabHC-ASM

SEQ ID NO: 115: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hARSA, IgG4HC-ARSA

SEQ ID NO: 116: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hARSA, IgG4HC-GS5-ARSA

SEQ ID NO: 117: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hARSA, IgG4HC-GS10-ARSA

SEQ ID NO: 118: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hARSA, IgG4HC-GS20-ARSA

SEQ ID NO: 119: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hARSA, FabHC-GS3-ARSA

SEQ ID NO: 120: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hARSA, FabHC-GS5-ARSA

SEQ ID NO: 121: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hARSA, FabHC-GS10-ARSA

SEQ ID NO: 122: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hARSA, FabHC-GS20-ARSA

SEQ ID NO: 123: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hARSA, Tandem FabHC-ARSA

SEQ ID NO: 124: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hSGSH, IgG4HC-SGSH

SEQ ID NO: 125: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hSGSH, IgG4HC-GS5-SGSH

SEQ ID NO: 126: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hSGSH, IgG4HC-GS10-SGSH

SEQ ID NO: 127: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hSGSH, IgG4HC-GS20-SGSH

SEQ ID NO: 128: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hSGSH, FabHC-GS3-SGSH

SEQ ID NO: 129: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hSGSH, Tandem FabHC-SGSH

SEQ ID NO: 130: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGBA, IgG4HC-GBA

SEQ ID NO: 131: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGBA, IgG4HC-GS5-GBA

SEQ ID NO: 132: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGBA, IgG4HC-GS10-GBA

SEQ ID NO: 133: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGBA, IgG4HC-GS20-GBA

SEQ ID NO: 134: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGBA, FabHC-GS3-GBA

SEQ ID NO: 135: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGBA, Tandem FabHC-GBA

SEQ ID NO: 136: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hTPP-1, IgG4HC-TPP1

SEQ ID NO: 137:. Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hTPP-1, IgG4HC-GS5-TPP1

SEQ ID NO: 138: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hTPP-1, IgG4HC-GS 10-TPP1

SEQ ID NO: 139: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hTPP-1, IgG4HC-GS20-TPP1

SEQ ID NO: 140: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hTPP-1, FabHC-GS3-TPP1

SEQ ID NO: 141: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hTPP-1, TandemFab HC-TPP1

SEQ ID NO: 142: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hNAGLU, IgG4HC-NAGLU

SEQ ID NO: 143: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hNAGLU, IgG4HC-GS5-NAGLU

SEQ ID NO: 144: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hNAGLU, IgG4HC-GS10-NAGLU

SEQ ID NO: 145: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hNAGLU, IgG4HC-GS20-NAGLU

SEQ ID NO: 146: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hNAGLU, FabHC-GS3-NAGLU

SEQ ID NO: 147: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hNAGLU, FabHC-GS5-NAGLU

SEQ ID NO: 148: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hNAGLU, FabHC-GS10-NAGLU

SEQ ID NO: 149: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hNAGLU, FabHC-GS20-NAGLU

SEQ ID NO: 150: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGUSB, IgG4HC-GUSB

SEQ ID NO: 151: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGUSB, IgG4HC-GS5-GUSB

SEQ ID NO: 152: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGUSB, IgG4HC-GS10-GUSB

SEQ ID NO: 153: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGUSB, IgG4HC-GS20-GUSB

SEQ ID NO: 154: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGUSB, FabHC-G53-GUSB

SEQ ID NO: 155: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGUSB, FabHC-GS5-GUSB

SEQ ID NO: 156: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGUSB, FabHC-GS10-GUSB

SEQ ID NO: 157: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGUSB, FabHC-GS20-GUSB

SEQ ID NO: 158: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGALC, IgG4HC-GALC

SEQ ID NO: 159: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGALC, IgG4HC-GS5-GALC

SEQ ID NO: 160: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGALC, IgG4HC-GS10-GALC

SEQ ID NO: 161: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hGALC, IgG4HC-GS20-GALC

SEQ ID NO: 162: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGALC, FabHC-GS3-GALC

SEQ ID NO: 163: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGALC, FabHC-GS5-GALC

SEQ ID NO: 164: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGALC, FabHC-GS10-GALC

SEQ ID NO: 165: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hGALC, FabHC-GS20-GALC

SEQ ID NO: 166: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hAC, IgG4 HC-AC

SEQ ID NO: 167: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hAC, IgG4HC-GS5-AC

SEQ ID NO: 168: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hAC, IgG4HC-GS10-AC

SEQ ID NO: 169: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hAC, IgG4HC-GS20-AC

SEQ ID NO: 170: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hAC,

Fab HC-GS3-AC

SEQ ID NO: 171: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hAC, FabHC-GS5-AC

SEQ ID NO: 172: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hAC, Fab HC-GS10-AC

SEQ ID NO: 173: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hAC, FabHC-GS20-AC

SEQ ID NO: 174: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hFUCA1, IgG4HC-FUCA1

SEQ ID NO: 175: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hFUCA1, IgG4HC-GS5-FUCA1

SEQ ID NO: 176: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hFUCA1, IgG4HC-GS10-FUCA1

SEQ ID NO: 177: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hFUCA1, IgG4HC-GS20-FUCA1

SEQ ID NO: 178: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hFUCA1, FabHC-GS3-FUCA1

SEQ ID NO: 179: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hFUCA1, FabHC-GS5-FUCA1

SEQ ID NO: 180: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hFUCA1, FabHC-GS10-FUCA1

SEQ ID NO: 181: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hFUCA1, FabHC-GS20-FUCA1

SEQ ID NO: 182: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hLAMAN, IgG4HC-LAMAN

SEQ ID NO: 183: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hLAMAN, IgG4HC-GS5-LAMAN

SEQ ID NO: 184: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hLAMAN, IgG4HC-GS10-LAMAN

SEQ ID NO: 185: Amino acid sequence of fusion protein between the heavychain (IgG4) of humanized anti-hTfR antibody No. 3N and hLAMAN, IgG4HC-GS20-LAMAN

SEQ ID NO: 186: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hLAMAN, FabHC-GS3-LAMAN

SEQ ID NO: 187: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hLAMAN, FabHC-GS5-LAMAN

SEQ ID NO: 188: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hLAMAN, FabHC-GS10-LAMAN

SEQ ID NO: 189: Amino acid sequence of fusion protein between the Fabheavy chain of humanized anti-hTfR antibody No. 3N and hLAMAN, FabHC-GS20-LAMAN

1. An anti-human transferrin receptor antibody, wherein in the heavychain variable region of the antibody, wherein (a) CDR1 comprises theamino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, (b) CDR2comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or SEQ IDNO:
 16. 2-79. (canceled)
 80. The antibody according to claim 1, whereinthe framework region 3 of the heavy chain comprises the amino acidsequence of SEQ ID NO:
 64. 81. The antibody according to claim 1,wherein in the heavy chain variable region, the CDR2 comprises an aminoacid sequence having sequence identity not lower than 80% to the aminoacid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, in place thereof, andthe CDR3 comprises an amino acid sequence having sequence identity notlower than 80% to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO:16, in place thereof.
 82. The antibody according to claim 1, wherein inthe heavy chain variable region, the CDR2 comprises an amino acidsequence having sequence identity not lower than 90% to the amino acidsequence of SEQ ID NO: 13 or SEQ ID NO: 14, in place thereof, and theCDR3 comprises an amino acid sequence having sequence identity not lowerthan 90% to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16,in place thereof.
 83. The antibody according to claim 1, wherein theheavy chain variable region comprises an amino acid sequence modifiedfrom at least one amino acid sequence of (a) SEQ ID NO: 62 or SEQ ID NO:63 as to the CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 as to the CDR2,(c) SEQ ID NO: 15 or SEQ ID NO: 16 as to the CDR3, and (d) SEQ ID NO: 64as to the framework region 3, by the substitution, deletion or additionof 1 to 5 amino acids, in place thereof, wherein in the CDR1, methioninepositioned at position 5 from the N-terminal side of the amino acidsequence of SEQ ID NO: 62 or SEQ ID NO: 63 is also located at the sameposition in the amino acid sequence thus modified, and in the frameworkregion 3, leucine positioned at position 17 from the N-terminal side ofSEQ ID NO: 64 is also located at the same position in the amino acidsequence thus modified.
 84. The antibody according to claim 1, whereinthe heavy chain variable region comprises an amino acid sequencemodified from at least one amino acid sequence of (a) SEQ ID NO: 62 orSEQ ID NO: 63 as to the CDR1, (b) SEQ ID NO: 13 or SEQ ID NO: 14 as tothe CDR2, (c) SEQ ID NO: 15 or SEQ ID NO: 16 as to the CDR3, and (d) SEQID NO: 64 as to the framework region 3, by the substitution, deletion oraddition of 1 to 3 amino acids, in place thereof, wherein in the CDR1,methionine positioned at position 5 from the N-terminal side of theamino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63 is also located atthe same position in the amino acid sequence thus modified, and in theframework region 3, leucine positioned at position 17 from theN-terminal side of SEQ ID NO: 64 is also located at the same position inthe amino acid sequence thus modified.
 85. The antibody according toclaim 80, wherein the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:
 65. 86. The antibody according to claim 85,wherein the heavy chain variable region comprises, in a portion exceptfor the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 of theCDR1 and SEQ ID NO: 64 of the framework region 3, an amino acid sequencehaving sequence identity not lower than 80% to the portion in place ofthe portion.
 87. The antibody according to claim 85, wherein the heavychain variable region comprises, in a portion except for the amino acidsequences of SEQ ID NO: 62 or SEQ ID NO: 63 of the CDR1 and SEQ ID NO:64 of the framework region 3, an amino acid sequence having sequenceidentity not lower than 90% to the portion in place of the portion. 88.The antibody according to claim 85, wherein the antibody comprises anamino acid sequence modified from the amino acid sequence constitutingthe heavy chain variable region, by the substitution, deletion oraddition of 1 to 5 amino acids, in place thereof, wherein in the CDR1,methionine positioned at position 5 from the N-terminus of the aminoacid sequence of SEQ ID NO: 63 is also located at the same position inthe amino acid sequence thus modified, and in the framework region 3,leucine positioned at position 17 from the N-terminal side of SEQ ID NO:64 is also located at the same position in the amino acid sequence thusmodified.
 89. The antibody according to claim 85, wherein the antibodycomprises an amino acid sequence modified from the amino acid sequenceconstituting the heavy chain variable region, by the substitution,deletion or addition of 1 to 3 amino acids, in place thereof, wherein inthe CDR1, methionine positioned at position 5 from the N-terminus of theamino acid sequence of SEQ ID NO: 63 is also located at the sameposition in the amino acid sequence thus modified, and in the frameworkregion 3, leucine positioned at position 17 from the N-terminal side ofSEQ ID NO: 64 is also located at the same position in the amino acidsequence thus modified.
 90. The antibody according to claim 85, whereinthe heavy chain comprises the amino acid sequence of SEQ ID NO: 66 orSEQ ID NO:
 68. 91. The antibody according to claim 90, wherein the heavychain comprises an amino acid sequence having sequence identity notlower than 80% to a portion except for the amino acid sequences of SEQID NO: 62 or SEQ ID NO: 63 of the CDR1 and SEQ ID NO: 64 of theframework region 3, in place of the portion.
 92. The antibody accordingto claim 90, wherein the heavy chain comprises an amino acid sequencehaving sequence identity not lower than 90% to a portion except for theamino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 63 of the CDR1 andSEQ ID NO: 64 of the framework region 3, in place of the portion. 93.The antibody according to claim 90, wherein the antibody comprises anamino acid sequence modified from the amino acid sequence constitutingthe heavy chain, by the substitution, deletion or addition of 1 to 5amino acids, in place thereof, wherein in the CDR1, methioninepositioned at position 5 from the N-terminus of the amino acid sequenceof SEQ ID NO: 63 is also located at the same position in the amino acidsequence thus modified, and in the framework region 3, leucinepositioned at position 17 from the N-terminal side of SEQ ID NO: 64 isalso located at the same position in the amino acid sequence thusmodified.
 94. The antibody according to claim 90, wherein the antibodycomprises an amino acid sequence modified from the amino acid sequenceconstituting the heavy chain, by the substitution, deletion or additionof 1 to 3 amino acids, in place thereof, wherein in the CDR1, methioninepositioned at position 5 from the N-terminus of the amino acid sequenceof SEQ ID NO: 63 is also located at the same position in the amino acidsequence thus modified, and in the framework region 3, leucinepositioned at position 17 from the N-terminal side of SEQ ID NO: 64 isalso located at the same position in the amino acid sequence thusmodified.
 95. The antibody according to claim 1, wherein in the lightchain variable region of the antibody, (a) CDR1 comprises the amino acidsequence of SEQ ID NO: 6 or SEQ ID NO: 7, (b) CDR2 comprises the aminoacid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acidsequence Lys-Val-Ser, and (c) CDR3 comprises the amino acid sequence ofSEQ ID NO:
 10. 96. The antibody according to claim 95, wherein in thelight chain variable region, (a) the CDR1 comprises an amino acidsequence having sequence identity not lower than 80% to the amino acidsequence of SEQ ID NO: 6 or SEQ ID NO: 7, in place thereof, (b) the CDR2comprises an amino acid sequence having sequence identity not lower than80% to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or theamino acid sequence Lys-Val-Ser, in place thereof, and (c) the CDR3comprises an amino acid sequence having sequence identity not lower than80% to the amino acid sequence set forth as SEQ ID NO: 10, in placethereof.
 97. The antibody according to claim 95, wherein in the lightchain variable region, (a) the CDR1 comprises an amino acid sequencehaving sequence identity not lower than 90% to the amino acid sequenceof SEQ ID NO: 6 or SEQ ID NO: 7, in place thereof, (b) the CDR2comprises an amino acid sequence having sequence identity not lower than90% to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or theamino acid sequence Lys-Val-Ser, in place thereof, and (c) the CDR3comprises an amino acid sequence having sequence identity not lower than90% to the amino acid sequence of SEQ ID NO: 10, in place thereof. 98.The antibody according to claim 95, wherein the light chain variableregion comprises an amino acid sequence modified from at least one aminoacid sequence of (a) SEQ ID NO: 6 or SEQ ID NO: 7 as to the CDR1, (b)SEQ ID NO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser asto the CDR2, and (c) SEQ ID NO: 10 as to the CDR3, by the substitution,deletion or addition of 1 to 5 amino acids, in place thereof.
 99. Theantibody according to claim 95, wherein the light chain variable regioncomprises an amino acid sequence modified from at least one amino acidsequence of (a) SEQ ID NO: 6 or SEQ ID NO: 7 as to the CDR1, (b) SEQ IDNO: 8 or SEQ ID NO: 9, or the amino acid sequence Lys-Val-Ser as to theCDR2, and (c) SEQ ID NO: 10 as to the CDR3, by the substitution,deletion or addition of 1 to 3 amino acids, in place thereof.
 100. Theantibody according to claim 1, wherein the light chain variable regionof the antibody comprises the amino acid sequence of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.101. The antibody according to claim 100, wherein the antibody comprisesan amino acid sequence having sequence identity not lower than 80% tothe amino acid sequence of the light chain variable region, in placethereof.
 102. The antibody according to claim 100, wherein the antibodycomprises an amino acid sequence having a sequence identity not lowerthan 90% to the amino acid sequence of the light chain variable region,in place thereof.
 103. The antibody according to claim 100, wherein theantibody comprises an amino acid sequence modified from the amino acidsequence of the light chain variable region by the substitution,deletion or addition of 1 to 5 amino acids, in place thereof.
 104. Theantibody according to claim 100, wherein the antibody comprises an aminoacid sequence modified from the amino acid sequence of the light chainvariable region by the substitution, deletion or addition of 1 to 3amino acids, in place thereof.
 105. The antibody according to claim 1,wherein the light chain of the antibody comprises the amino acidsequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO:29.
 106. The antibody according to claim 105, wherein the antibodycomprises an amino acid sequence having sequence identity not lower than80% to the amino acid sequence of the light chain, in place thereof.107. The antibody according to claim 105, wherein the antibody comprisesan amino acid sequence having sequence identity not lower than 90% tothe amino acid sequence of the light chain, in place thereof.
 108. Theantibody according to claim 105, wherein the antibody comprises an aminoacid sequence modified from the amino acid sequence of the light chainby the substitution, deletion or addition of 1 to 5 amino acids, inplace thereof.
 109. The antibody according to claim 105, wherein theantibody comprises an amino acid sequence modified from the amino acidsequence of the light chain by the substitution, deletion or addition of1 to 3 amino acids, in place thereof.
 110. The antibody according toclaim 1, wherein the antibody has an affinity to both the extracellularregion of human transferrin receptor and the extracellular region ofmonkey transferrin receptor.
 111. The antibody according to claim 110,wherein the dissociation constant of its complex with the extracellularregion of human transferrin receptor is not greater than 1×10⁻¹⁰ M, andthe dissociation constant of its complex with the extracellular regionof monkey transferrin receptor is not greater than 1×10⁻⁹ M.
 112. Theantibody according to claim 1, wherein the antibody is Fab antibody,F(ab′)₂ antibody, or F(ab′) antibody.
 113. The anti-human transferrinreceptor antibody according to claim 1, wherein the antibody is asingle-chain antibody selected from the group consisting of scFab,scF(ab′), scF(ab′)₂ and scFv.
 114. The antibody according to claim 113,wherein the light chain and the heavy chain thereof are linked via alinker sequence.
 115. The antibody according to claim 114, wherein theheavy chain is linked, via a linker sequence, to the light chain on theC-terminal side thereof.
 116. The antibody according to claim 113,wherein the light chain is linked, via a linker sequence, to the heavychain on the C-terminal side thereof.
 117. The antibody according toclaim 114, wherein the linker sequence consists of 8 to 50 amino acidresidues.
 118. The antibody according to claim 117, wherein the linkersequence is selected from the group consisting of the amino acidsequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acidsequence Gly-Gly-Gly, the amino acid sequences set forth as SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NO: 5, the amino acid sequence consisting ofthree consecutively linked amino acid sequences each set forth as SEQ IDNO: 3, and the amino acid sequences consisting of 1 to 10 thereof thatare consecutively linked.
 119. A fusion protein comprising an anti-humantransferrin receptor antibody and a different protein (A), wherein theanti-human transferrin receptor antibody is the antibody according toclaim 1, and wherein the protein (A) is linked to the light chain of theantibody on the C-terminal side or the N-terminal side thereof.
 120. Thefusion protein according to claim 119, wherein the protein (A) islinked, directly or via a linker, to the light chain on the C-terminalside or the N-terminal side thereof.
 121. The fusion protein accordingto claim 119, wherein the protein (A) is linked, via a linker, to thelight chain on the C-terminal side or the N-terminal side thereof. 122.The fusion protein according to claim 121, wherein the linker is apeptide consisting of 1 to 50 amino acid residues.
 123. The fusionprotein according to claim 122, wherein the linker is a peptidecomprising an amino acid sequence selected from the group consisting ofa single glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO:3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence ofSEQ ID NO: 5, and the amino acid sequences consisting of 1 to 10 thereofthat are consecutively linked.
 124. A fusion protein of an anti-humantransferrin receptor antibody and a different protein (A), wherein theanti-human transferrin receptor antibody is the antibody according toclaim 1, and the protein (A) is linked to the heavy chain of theantibody on the C-terminal side or the N-terminal side thereof.
 125. Thefusion protein according to claim 124, wherein the protein (A) islinked, directly or via a linker, to the heavy chain on the C-terminalside or the N-terminal side thereof.
 126. The fusion protein accordingto claim 124, wherein the protein (A) is linked, via a linker, to theheavy chain on the C-terminal side or the N-terminal side thereof. 127.The fusion protein according to claim 126, wherein the linker sequenceis a peptide consisting of 1 to 50 amino acid residues.
 128. The fusionprotein according to claim 127, wherein the linker is a peptidecomprising an amino acid sequence selected from the group consisting ofa single glycine, a single serine, the amino acid sequence Gly-Ser, theamino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO:3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence ofSEQ ID NO: 5, and the amino acid sequences consisting of 1 to 10 thereofthat are consecutively linked.
 129. The fusion protein according toclaim 119, wherein the protein (A) is a protein originating from human.130. The fusion protein according to claim 119, wherein the protein (A)is selected from the group consisting of nerve growth factor (NGF),lysosomal enzymes, ciliary neurotrophic factor (CNTF), glial cellline-derived neurotrophic factor (GDNF), neurotrophin-3,neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin,darbepoetin, activin, basic fibroblast growth factor (bFGF), fibroblastgrowth factor 2 (FGF2), epidermal growth factor (EGF), vascularendothelial growth factor (VEGF), interferon α, interferon β, interferonγ, interleukin 6, granulocyte-macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), cytokines, tumor necrosis factor αreceptor (TNF-α receptor), PD-1 ligands, PD-L1, PD-L2 enzymes havingβ-amyloid-degrading activity, anti-β-amyloid antibody, anti-BACEantibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody,anti-PD-L2 antibody, anti-HER2 antibody, anti-TNF-α antibody,anti-CTLA-4 antibody, and other antibody medicines.
 131. The fusionprotein according to claim 119, wherein the protein (A) is a lysosomalenzyme, wherein the lysosomal enzyme is selected from the groupconsisting of α-L-iduronidase, iduronate 2-sulfatase, human acidicα-glucosidase, glucocerebrosidase, β-galactosidase, GM2 activatorprotein, β-hexosaminidase A, β-hexosaminidase B,N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase,galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase,aspartylglucosaminidase, α-N-acetylgalactosaminidase, acidicsphingomyelinase, α-galactosidase A, β-glucuronidase, heparanN-sulfatase, α-N-acetylglucosaminidase, acetyl CoA:α-α-glucosaminideN-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acidceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl proteinthioesterase 1, tripeptidyl-peptidase 1, hyaluronidase 1, CLN1 and CLN2.132. The fusion protein according to claim 119, wherein the protein (A)is human iduronate 2-sulfatase, human acidic α-glucosidase, or humanα-L-iduronidase.
 133. The fusion protein according to claim 129, whereinthe protein (A) is human acidic α-glucosidase, wherein (1) the lightchain of the antibody comprises the amino acid sequence of SEQ ID NO:23, and (2) the heavy chain of the antibody is linked, on the C-terminalside thereof and via the amino acid sequence Gly-Ser, to the humanacidic α-glucosidase, thereby forming the amino acid sequence of SEQ IDNO: 57 or SEQ ID NO:
 58. 134. The fusion protein according to claim 129,wherein the protein (A) is human acidic α-glucosidase, wherein (1) thelight chain of the antibody comprises the amino acid sequence of SEQ IDNO: 23, and (2) the heavy chain of the antibody comprises the amino acidsequence of SEQ ID NO: 66 or SEQ ID NO: 68, and the heavy chain islinked, on the C-terminal side thereof and via the amino acid sequenceGly-Ser, to the human acidic α-glucosidase having the amino acidsequence of SEQ ID NO: 55 or
 56. 135. The fusion protein according toclaim 129, wherein the protein (A) is human acidic α-glucosidase,wherein the antibody is Fab antibody, and wherein (1) the light chain ofthe antibody comprises the amino acid sequence of SEQ ID NO: 23, and (2)the heavy chain of the antibody is linked, on the C-terminal sidethereof and via the amino acid sequence consisting of threeconsecutively linked amino acid sequences each of SEQ ID NO: 3, to thehuman acidic α-glucosidase, thereby forming the amino acid sequence ofSEQ ID NO:
 89. 136. The fusion protein according to claim 129, whereinthe protein (A) is human acidic α-glucosidase, wherein the antibody isFab antibody, and wherein (1) the light chain of the antibody comprisesthe amino acid sequence of SEQ ID NO: 23, and (2) the heavy chain of theantibody comprises the amino acid sequence of SEQ ID NO: 61, and theheavy chain is linked, on the C-terminal side thereof and via the aminoacid sequence consisting of three consecutively linked amino acidsequences each of SEQ ID NO: 3, to the human acidic α-glucosidase havingthe amino acid sequence of SEQ ID NO: 55 or
 56. 137. The fusion proteinaccording to claim 129, wherein the protein (A) is humanα-L-iduronidase, wherein the antibody is Fab antibody, and wherein (1)the light chain of the antibody comprises the amino acid sequence of SEQID NO: 23, and (2) the heavy chain of the antibody is linked, on theC-terminal side thereof and via the amino acid sequence consisting ofthree consecutively linked amino acid sequences each of SEQ ID NO: 3, tothe human α-L-iduronidase, thereby forming the amino acid sequence ofSEQ ID NO:
 93. 138. The fusion protein according to claim 129, whereinthe protein (A) is human α-L-iduronidase, wherein the antibody is Fabantibody, and wherein (1) the light chain of the antibody comprises theamino acid sequence of SEQ ID NO: 23, and (2) the heavy chain of theantibody comprises the amino acid sequence of SEQ ID NO: 61, and theheavy chain is linked, on the C-terminal side thereof and via the aminoacid sequence consisting of three consecutively linked amino acidsequences of SEQ ID NO: 3, to the human α-L-iduronidase having the aminoacid sequence of SEQ ID NO: 75 or
 76. 139. The fusion protein accordingto claim 119, wherein a human IgG Fc region or part thereof isintroduced between the protein (A) and the antibody.
 140. The fusionprotein according to claim 139, wherein the human IgG Fc region islinked, directly or via a linker sequence, to the protein (A) on theC-terminal side thereof, and the heavy chain or the light chain of theantibody is linked, directly or via a linker sequence, to the human IgGFc region on the C-terminal side thereof.
 141. The fusion proteinaccording to claim 139, wherein the human IgG Fc region comprises theamino acid sequence of SEQ ID NO:
 70. 142. The fusion protein accordingto claim 141, wherein the human IgG Fc region is linked, via a linkersequence, to a Fab heavy chain consisting of the amino acid sequence ofSEQ ID NO: 61, and comprises the amino acid sequence of SEQ ID NO: 71formed thereby.
 143. A DNA fragment encoding the amino acid sequence ofthe anti-human transferrin receptor antibody according to claim
 1. 144.A DNA fragment encoding the amino acid sequence of the fusion proteinaccording to claim
 119. 145. An expression vector comprising the DNAfragment according to claim
 143. 146. A mammalian cell transformed withthe expression vector according to claim
 145. 147. An anti-humantransferrin receptor antibody-pharmacologically active compound complex,wherein the light chain and/or the heavy chain of the anti-humantransferrin receptor antibody according to claim 1 is linked to alow-molecular-weight pharmacologically active compound that needs to beallowed to pass through the blood-brain barrier and exhibit the functionthereof in the brain.
 148. The anti-human transferrin receptorantibody-pharmacologically active compound complex according to claim147, wherein the pharmacologically active compound is any one selectedfrom the group consisting of anticancer drug, therapeutic agent forAlzheimer's disease, therapeutic agent for Parkinson's disease,therapeutic agent for Huntington's disease, therapeutic agent forschizophrenia, antidepressant, therapeutic agent for multiple sclerosis,therapeutic agent for amyotrophic lateral sclerosis, therapeutic agentfor tumors of central nervous system including brain tumor, therapeuticagent for lysosomal storage disease accompanied by encephalopathy,therapeutic agent for glycogenosis, therapeutic agent for musculardystrophy, therapeutic agent for cerebral ischemia, therapeutic agentfor prion diseases, therapeutic agent for traumatic central nervoussystem disorders, therapeutic agent for viral and bacterial centralnervous system diseases, pharmaceutical agent used for recovery afterbrain surgery, pharmaceutical agent used for recovery after spinalsurgery, siRNA, antisense DNA, and peptide.
 149. A method for treatmentof a disorder of the central nervous system comprising parenterallyadministering to a patient with the disorder a therapeutically effectiveamount of the physiologically active protein, or pharmacologicallyactive low-molecular-weight compound, for the disorder, in the form of aconjugate with the molecule of the anti-human transferrin receptorantibody according to claim
 1. 150. A method for the treatment of adisease of the central nervous system accompanying Pompe's diseasecomprising parenterally administering a therapeutically effective amountof the fusion protein according to claim 130 to a patient with thedisease.
 151. A method for the treatment of a disease of the centralnervous system accompanying Hurler syndrome or Hurler-Scheie syndrome,the method comprising parenterally administering a therapeuticallyeffective amount of the fusion protein according to claim 130 to apatient with the disease.
 152. A method for the production of a fusionprotein according to claim 119, comprising a step of culturing mammaliancells transformed with an expression vector comprising a DNA fragmentthat is incorporated therein encoding the amino acid sequence of thefusion protein to let the mammalian cells secrete the fusion protein inthe culture medium.
 153. A method for the production of a fusion proteinaccording to claim 124, comprising a step of culturing mammalian cellstransformed with an expression vector comprising a DNA fragment that isincorporated therein encoding the amino acid sequence of the fusionprotein to let the mammalian cells secrete the fusion protein in theculture medium.